Solution-based methods for rna expression profiling

ABSTRACT

The present invention is directed to novel high-throughput, low-cost, and flexible solution-based methods for RNA expression profiling, including expression of microRNAs and mRNAs.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of U.S. Utility application Ser. No. 12/870,126, filed Aug. 27, 2010, now abandoned, which is a Continuation Application of U.S. Utility application Ser. No. 11/449,155, filed Jun. 8, 2006, now abandoned, which claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No. 60/689,110 filed Jun. 8, 2005, the contents of which are herein incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 28, 2013 is named SEQ_(—)037822-056701-C.txt and is 202,138 bytes in size.

FIELD OF THE INVENTION

The present invention is directed to methods of screening for malignancies, cellular disorders, and other physiological states as well as novel high-throughput, low-cost, and flexible solution-based methods for RNA expression profiling, including expression of microRNAs and mRNAs.

BACKGROUND OF THE INVENTION

The availability of high-performance RNA profiling technologies is central to the elucidation of the mechanisms of action of disease genes and the identification of small molecule therapeutics by molecular signature screening (Lamb et al., Cell 114:323-34 (2003); Stegmaier et al., Nature Genetics 36:257-63 (2004)). For example, detection and quantification of differentially expressed genes in a number of conditions including malignancy, cellular disorders, etc. would be useful in the diagnosis, prognosis and treatment of these pathological conditions. Quantification of gene expression would also be useful in indicating susceptibility to a range of conditions and following up effects of pharmaceuticals or toxins on molecular level. These methods can also be used to screen for molecules that provide a desired gene profile.

The power of being able to simultaneously measure the expression level of multiple mRNA species has been of recent interest. For example, the expression of seventy and eighty-one transcripts have together been shown to outperform established clinical and histologic parameters in disease outcome prediction for breast cancer (van de Vijver et al., New Eng. J. Med. 347: 1999-2009 (2002)) and follicular lymphoma (Glas et al., Blood 105:301-7 (2005)), respectively.

MicroRNAs are thought to act as post-transcriptional modulators of gene expression, and have been implicated as regulators of developmental timing, neuronal differentiation, cell proliferation, programmed cell death, and fat metabolism. Determining expression profiles of microRNAs is particularly challenging however because of their short size, typically around 21 base pairs, and high degree of sequence homology, where different microRNAs may differ by only a single base pair. It would also be highly desirable to simultaneously measure the expression level of microRNAs, a recently identified class of small non-coding RNA species.

The rapid pace of discovery of new genes generated by large-scale genomic and proteomic initiatives has required the development of high-throughput strategies to quantify the expression of a large number of genes and their alternatively spliced isoforms, as well as elucidate their biological functions, regulations and interactions. (Consortium, E. P. (2004) Science 306, 636-40; Lander et al., Nature 409, 860-921 (2001)) A number of high-throughput techniques have been developed to detect and quantify nucleic acids. Microarray-based analysis has been one widely used high-throughput technique used to study nucleic acids. Another approach for high-throughput analysis of nucleic acids involves the sequencing of a short tag of each transcript, including expressed sequence tag (EST) sequencing (Lander et al., 2001) and serial analysis of gene expression (SAGE) (Velculescu et al., Science 270, 484-7 (1995)).

However, both microarray and tag-sequencing techniques are associated with a number of significant problems. These techniques typically are not sufficiently sensitive and demand relatively high input levels of mRNA that are often unavailable, particularly when studying human diseases. In addition, the array quality is often a problem for cDNA or oligonucleotide microarrays. For example, most researchers cannot confirm the identity of what is immobilized on the surface of a microarray and generally have limited capacity to check and control possible errors in the microarray fabrication. Additionally, the high costs of microarrays have caused many investigators to perform relatively few control experiments to assess the reliability, validity, and repeatability of their findings. Moreover, given the high costs of microarray fabrication, custom designing arrays to tailor analysis to an individual expression profile is simply impractical in many instances. For the tag-sequencing analysis, a large amount of sequencing effort, generally slow and costly, is needed for tag-based analysis and the sensitivity of tag-based analyses is relatively low and high sensitivity can only be achieved by sequencing a large number of tag sequences.

Thus it would be desirable to develop simple, flexible, low-cost, high-throughput methods for the sensitive and accurate quantification of nucleic acids, which can be easily automated and scaled up to accommodate testing of large numbers of samples and overcome the problems associated with available techniques. Such a method would permit diagnostic, prognostic and therapeutic purposes, and would facilitate genomic, pharmacogenomic and proteomic applications, including the discovery of small molecule therapeutics.

SUMMARY OF THE INVENTION

We have now discovered simple, flexible, low-cost and high-throughput solution-based methods for expression profiling nucleic acids. More specifically, the invention provides methods for detection of multiple genes in a single reaction, including for the detection of mRNAs and microRNAs.

The present invention provides a solution-based method for determining the expression level of a population of target nucleic acids, by a) providing in solution a population of target-specific bead sets, where each target-specific bead set is individually detectable and comprises a capture probe which corresponds to an individual target nucleic acid, referred to as an individual bead set; b) hybridizing in solution the population of target-specific bead sets with a population of molecules that can contain a population of detectable target molecules, where each target nucleic acid has been transformed into a corresponding detectable target molecule which will specifically bind to its corresponding individual target-specific bead set; and c) screening in solution for detectable target molecules hybridized to target-specific beads to determine the expression level of the population of target nucleic acids.

In one embodiment, the target-specific bead sets can have at least 5 individual bead sets that can bind with a corresponding set of target nucleic acids. The population of target-specific beads can contain at least 100 individual bead sets that bind with a corresponding set of target nucleic acids.

One preferred embodiment provides a method for detection of populations of mRNAs. In this method, mRNA is transformed into a corresponding detectable target molecule by a) reverse transcribing the mRNA to generate a cDNA; b) hybridizing an upstream probe and a downstream probe to the cDNA, where the upstream probe has a universal upstream sequence and an upstream target-specific sequence, and the downstream probe has a universal downstream sequence and a downstream target-specific sequence, such that when the upstream probe and the downstream probe are both hybridized to the cDNA the two probes are capable of being ligated; c) ligating the two probes to generate ligation complexes; and d) amplifying the ligation complexes with a universal upstream primer and a universal downstream primer, which are complementary to the universal upstream sequence and the universal downstream sequence, respectively. In this method, at least one of universal primers is detectably labeled, such that product of the amplification is detectably labeled, thereby generating a detectable target molecule which corresponds to the target nucleic acid. In this method, either the upstream probe or the downstream probe also has an amplicon tag between the universal sequence and the target-specific. The amplicon tag has a nucleic acid sequence that is unique for the mRNA to be detected, and that is complementary to the sequence of the capture probe of the corresponding bead set, allowing the detectable nucleic acid molecule to hybridize to the bead set with the complementary capture probe.

One embodiment of the invention provides the use of these multiplex mRNA detection methods to screen for the presence of a particular physiological state in a test sample, such as a malignancy, infection or a cellular disorder. In one embodiment, the genes which are specifically associated with one physiological state but not another physiological state are already determined; such a group of genes is typically referred to as an expression signature. To screen for a physiological state using the mRNA detection methods, one first determines the expression signature of a group of genes in the test sample; and then compares the expression signature between the test sample and a corresponding control sample, where a difference in the expression signature between the test sample and the control sample is indicative of the test sample comprising said malignant cells, infected cells or cellular disorder. In one embodiment, the expression signature has at least 5 genes.

One embodiment of the invention provides a method for identifying an expression signature for a physiological state, using the multiplex mRNA detection methods to rapidly screen for genes which are differentially expressed between two physiological states. In one embodiment, the expression signature has at least 5 genes. Examples of physiological states include the presence of a cancer, infection, or a cellular disorder. To identify novel expression signatures, one isolates cells from two groups of individuals, one with and one without the physiological state of interest, and then identifies those genes which are differentially expressed in the two groups of individuals. For those genes which differ at a statistically significant level, linear regression analysis can be applied to identify an expression signature of a gene group that is indicative of an individual having the physiological state of interest.

One preferred embodiment provides a method to detection of populations of microRNAs. In this method, microRNAs are transformed into corresponding detectable target molecules by first ligating at least one adaptor to each microRNA, generating an adaptor-microRNA molecule; and then detectably labeling the adaptor-microRNA molecule, thereby generating a detectable target molecule which corresponds to the target nucleic acid. In one embodiment, the adaptor-microRNA is detectably labeled by reverse transcription using the adaptor-microRNA as a template for polymerase chain reaction, wherein a pair of primers is used in said polymerase chain reaction, and wherein at least one of said primers is detectably labeled. In this method, the capture probe of the bead set which corresponds to an individual microRNA has a sequence which is complementary to the miRNA sequence, allowing the detectable target molecule to bind to the corresponding bead set.

The invention also provides the use of the multiplex microRNA detection methods to screen for the presence of a malignancy in a test sample. In one embodiment, one analyzes the level of expression of microRNAs in a test sample and a corresponding control sample, where a lower level of expression of microRNAs in the test sample relative to the control sample is indicative of the test sample containing malignant cells.

One embodiment of the invention provides a method of screening an individual at risk for cancer by obtaining at least two cell samples from the individual at different times; and determining the level of expression of microRNAs in the cell samples, where a lower level of expression of microRNAs in the later obtained cell sample compared to the earlier obtained cell sample is indicative of the individual being at risk for cancer.

Another embodiment of the invention provides methods of screening an individual at risk for cancer, by determining the level of expression for a specific group of microRNAs, sometimes referred to as a profile group of microRNAs, where lower expression of the profile group of microRNAs is associated with risk for a particular type of cancer.

One embodiment of the invention provides a method for identifying an active compound. In this embodiment, cells are contacted with a plurality of molecules including chemical compounds and biologic molecules, and the expression of a set of marker genes present in the cells is determined using the novel detection methods of the invention. To identify active compounds, the expression of the marker genes to identify a cellular phenotype is scored, the presence of a specific cellular phenotype being indicative of an active compound. In one embodiment the plurality of chemical compounds is a set of compounds selected from the group consisting of small molecule libraries, FDA approved drugs, synthetic chemical libraries, phage display libraries, dosage libraries. In another embodiment the active compound is an anti-cancer drug. In a further embodiment the active compound is a cellular differentiation factor. In certain embodiments, the set of marker genes can include genes encoding mRNAs and/or genes encoding microRNAs.

Another embodiment of the invention provides kits for determining in solution the expression level of a population of target nucleic acids. Kits can include a population of detectable bead sets, wherein each target-specific bead set is individually detectable and is capable of being coupled to a capture probe which corresponds to an individual target nucleic acid of interest; components for transforming a target nucleic acid of interest into a corresponding detectable target molecule which will specifically bind to its corresponding individual target-specific bead set; and instructions for performing the solution-based detection methods of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows one embodiment of the present method for multiplex detection of mRNAs. Transcripts are captured on immobilized poly-dT and reverse transcribed. Two oligonucleotide probes are designed against each transcript of interest. For example, the upstream probes contain in the embodiment illustrated 20 nt complementary to a universal primer (T7) site, one of one hundred different 24 nt FlexMAP barcodes, and a 20 nt sequence complementary to the 3′-end of the corresponding first-strand cDNA. The downstream probes are 5′-phosphorylated and contain a 20 nt sequence contiguous with the gene-specific fragment of the upstream probe and a 20 nt universal primer (T3) site. Probes are annealed to their targets, free probes removed, and juxtaposed probes joined by the action of Tag ligase to yield synthetic 104 nt amplification templates. PCR is performed with T3 and 5′-biotinylated T7 primers. Biotinylated barcoded amplicons are hybridized against a pool of one hundred sets of fluorescent microspheres each expressing capture probes complementary to one of the barcodes, and incubated with streptavidin-phycoerythrin (SA-PE) to fluorescently label biotin moieties. Captured labeled amplicons are quantified and beads decoded and counted by flow cytometry. This strategy is based on published methods (Elering et al., 2003; Yeakley et al., 2002).

FIG. 2 shows the reproducibility of an embodiment of the method. Mean expression levels for each transcript under each condition were computed and the deviation of each individual data point from its corresponding mean was recorded. A histogram of the fraction of data points in each of twelve bins of fold deviation values is shown. This plot represents 1,800 data points (two conditions×ninety transcripts×ten replicates).

FIG. 3 shows the results of comparison of expression levels in one embodiment. Plot of mean expression values reported by LMA-FlexMAP against IVT-GeneChip for each transcript under both conditions. Means were calculated as for FIG. 4.

FIG. 4 shows performance in a representative gene space. Total RNA from HL60 cells treated with tretinoin or vehicle (DMSO) alone were analyzed by LMA-FlexMAP in the space of ninety transcripts selected from IVT-GeneChip analysis of the same material. Plots depict log ratios of expression levels (tretinoin/DMSO) reported by both platforms for each transcript, in each of nine classes. Correlation coefficients of the log ratios between platforms within each class are shown. IVT-GeneChip, green bars; LMA-FlexMAP, yellow bars. Asterisks (*) flag failed features. Ratios were computed on the means of three parallel hybridizations of the pooled product from three amplification and labeling reactions (IVT-GeneChip) or ten parallel amplification and hybridization procedures (LMA-FlexMAP) for each condition. Basal expression categories are 20-60 (low), 60-125 (moderate) and >125 (high). Differential expression categories are 1.5-2.5× (low), 3-4.5× (moderate) and >5× (high).

FIGS. 5A-5B show schematics of target preparation and bead detection of miRNAs. (FIG. 5A) 18 to 26-nucleotide (nt) small RNAs were purified by denaturing PAGE (polyacrylamide gel electrophoresis) from total RNAs extracted from tissues or cells. Small RNAs underwent two steps of adaptor ligation utilizing both the 5′-phosphate and 3′-hydroxl groups, each followed by a denaturing purification. Ligation products were reverse-transcribed (RT) and PCR amplified using a common set of primers, with biotinylation on the sense primer. (FIG. 5B) Denatured targets were hybridized to beads coupled with capture probes for miRNAs. After binding to streptavidin-phycoerythrin (SAPE), the beads went through a flow cytometer that has two lasers and is capable of detecting both the bead identity and fluorescence intensity on each bead.

FIGS. 6A-6C show the specificity and accuracy of bead-based miRNA detection. (FIG. 6 a) Synthetic oligonucleotides corresponding to let-7 family and mutants (see FIG. 11 for sequence similarity) were PCR-labelled and hybridized separately on beads and a glass-microarray. Synthetic targets indicated on horizontal axis, capture probes on vertical axis. Values represent proportion of signal relative to correct probe (set to 100%). (FIG. 6B) Cumulative cross-hybridization on capture probes. (FIG. 6C) Northern blot vs. bead detection (lanes 1-7: HEL, K562, TF-1, 293, MCF-7, PC-3, SKMEL-5). Bead results shown at left (averages from three (HEL, TF-1, 293, MCF-7, PC-3) or two (K562, SKMEL-5) independent experiments; error bars indicate standard deviation).

FIG. 7A-7C show hierarchical clustering of miRNA expression. (FIG. 7 a) miRNA profiles of 218 samples covering multiple tissues were clustered (average linkage, correlation similarity; samples are columns, miRNAs are rows). Samples of epithelial (EP) origin or derived from the gastrointestinal tract (GI) are indicated. Supplementary FIG. 4 shows more detail. (FIG. 7B) Clustering of 73 bone marrow samples from patients with ALL. Colored bars indicate the ALL subtypes. (FIG. 7C) Comparison of miRNA data and mRNA data. For 89 epithelial samples from (FIG. 7A) that had mRNA expression data, hierarchical clustering was performed. Samples of GI origin are shown in blue. GI-derived samples largely cluster together in the space of miRNA expression, but not by mRNA expression. Abbreviations: STOM: stomach; PAN: pancreas; KID: kidney; PROST: prostate; UT: uterus; MESO: mesothelioma; BRST: breast; FCC: follicular lymphoma; MF: mycosis fungoides; LVR: liver; BLDR: bladder; MELA: melanoma; TALL: T-cell ALL; BALL: B-cell ALL; LBL: diffuse large-B cell lymphoma; AML: acute myelogenous leukemia; HYPER 47-50: hyperdiploid with 47 to 50 chromosomes; HYPER >50: hyperdiploid with over 50 chromosomes; MLL: mixed lineage leukaemia; NORMP: normal ploidy. Further details in Example 3.

FIGS. 8A-8C show comparison between normal and tumor samples reveals global changes in miRNA expression. (FIG. 8A) Markers were selected to correlate with normal vs. tumor distinction. Heatmap of miRNA expression is shown, with miRNAs sorted according to the variance-fixed t-test score. (FIG. 8B) miRNA markers of normal (norm) vs. tumor distinction in human tissues from (FIG. 8A) applied to normal lungs and lung adenocarcinomas of KRasLA1 mice. A k-nearest neighbor (kNN) classifier based on human sample-derived markers yielded a perfect classification of the mouse samples (Euclidean distance, k=3). Mouse tumor T_MLUNG_(—)5 (3rd from right) was occasionally classified as normal with other kNN parameters (Supplementary Information). (FIG. 8C) HL-60 cells were treated with ATRA (+) or vehicle (−) for the indicated days. Heatmap of miRNA expression from a representative experiment is shown.

FIGS. 9A-9M show unsupervised analysis of miRNA expression data. miRNA profiling data of 218 samples covering multiple tissues and cancers were filtered, and centred centered and normalized for each feature. The data were then subjected to hierarchical clustering on both the samples (horizontally oriented) and the features (vertically oriented, with probe names on the left), with average-linkage and Pearson correlation as a similarity measure. Sample names (staggered) are indicated on the top and miRNA names on the left. Tissue types and malignancy status (MAL; N for normal, T for tumor and TCL for tumor cell line) are represented by colored bars. Samples that belong to the epithelial origin (EP) or derived from the gastrointestinal tract (GI) are also annotated below the dendrogram. STOM: stomach; PAN: pancreas; KID: kidney; PROST: prostate; UT: uterus; MESO: mesothelioma; BRST (breast); FCC: follicular lymphoma; MF: mycosis fungoides; COLON: colon; LVR: liver; BLDR: bladder; OVARY: ovary; Lung: lung; MELA: melanoma; BRAIN: brain; TALL: T-cell ALL; BALL: B-cell ALL; LBL: diffused large-B cell lymphoma; AML: acute myelogenous leukaemia.

FIG. 10 shows comparison of miRNA expression levels of poorly differentiated and more-differentiated tumors. Poorly differentiated tumors (PD) with primary origins from colon, ovary, lung, breast (BRST) or lymphnode (LBL) were compared to more-differentiated tumors (non-PD) of the corresponding tissue types in the miGCM collection. After filtering out non-detectible miRNAs, the remaining 173 features were centered and normalized for each tissue type separately to a mean of 0 and a standard deviation of 1. A heatmap of the data is shown. Samples with the same tissue type and PD status were sorted according to total miRNA expression readings, with higher expressing samples on the left. Features were sorted according to the variance-thresholded t-test score.

FIG. 11 shows specificity and accuracy of the bead-based miRNA detection platform, probe similarity (for FIG. 6). Eleven synthetic oligonucleotides corresponding to human let-7 family of miRNAs or mutants were PCR-labelled. Each of the labelled targets was split and hybridized separately on the bead platform and on a glass microarray. The synthetic targets are indicated on the horizontal axis, and the capture probes are indicated on the vertical axis. The similarity of the capture probes are measured by the differences in nucleotides (nt) and indicated by shades of blue.

FIGS. 12A-12B show noise and linearity of bead detection of miRNAs. (FIG. 12 a) The noise of target preparation and bead detection was analyzed. Multiple analyses of the same RNA samples were performed. Expression data were log2-transformed after thresholding at 1 to avoid negative numbers. The standard deviation (std) of each miRNA was plotted against the mean of that miRNA. Data were generated from independent labeling reactions and detections of five replicates of MCF-7, four replicates of PC-3, three replicates of HEL, three replicates of TF-1 and three replicates of 293 cell RNAs. Note that most miRNAs have a standard deviation below 0.75 when their mean is above 5 (in log2 scale). (FIG. 12 b) Linearity of target preparation and bead detection. miRNAs were labeled and profiled from HEL cell total RNA with different starting amounts (10 ug, 5 ug, 2 ug and 0.5 ug, respectively). Data are averages of duplicate determinations, measured in median fluorescence intensity (MFI). Each line connects the readings of one miRNA with different amounts of starting material.

FIG. 13 shows hierarchical clustering analyses of miRNA data and mRNA data. For 89 epithelial samples that had successful expression data of both miRNAs and mRNAs, hierarchical clustering was performed using average linkage and correlation similarity, after gene filtering. Filtering of miRNA data eliminates genes that do not have expression values above a minimum threshold in any sample (see Supplementary Methods for details). Three different filtering methods were used for mRNA data. The first method (mRNA filt-1) uses the same criteria as used for miRNA data, resulting in 14546 genes. The second method (mRNA filt-2) employed a variation filter as described (Ramaswamy et al., 2001), and resulted in 6621 genes. The third method (mRNA filt-3) focused on transcription factors that passed the above variation filter, ending with 220 genes. Samples of gastrointestinal tract (GI) or non-GI origins are indicated. Tissue type (TT) and malignancy status (MAL) for normal (N) or tumor (T) samples are also indicated. Note that the GI-derived samples largely cluster together in the space of miRNA expression, but not by mRNA expression. Abbreviations: PAN: pancreas; KID: kidney; PROST: prostate; UT: uterus; MESO: mesothelioma; BRST: breast; COLON: colon; BLDR: bladder; OVARY: ovary; Lung: lung; MELA: melanoma.

FIGS. 14A-14C show In vitro erythroid differentiation. Purified CD34⁺ cells from human umbilical cord blood were induced to differentiate along the erythroid lineage. (FIG. 14A) Total cell counts were determined every two days. Data are averages of cell counts from a triplicate experiment and error bars represent standard deviations. (FIG. 14B) Markers of erythroid differentiation, CD71 and Glycophorin A (GlyA), were determined using flow cytometry. Percentages of cells with negative (−), low, or positive (+) marker staining are plotted. (FIG. 14C) miRNA expression profiles of differentiating erythrocytes were determined on days indicated after induction. Data were log₂-transformed, averaged among successfully profiled same-day samples and normalized to a mean of 0 and a standard deviation of 1 for each miRNA. Data were then filtered to eliminate miRNAs that do not have expression values higher than a minimum cut-off (7.25 on log₂ scale) in any sample. A heatmap of miRNA expression is shown, with red color indicating higher expression and blue for lower expression. Data shown are from a representative differentiation experiment of two performed.

FIG. 15 shows comparison of miRNA expression levels with an mRNA signature of proliferation. A consensus set of mRNA transcripts that positively correlate with proliferation rate was assembled based on published data (see Supplementary Data). Data for miRNA and mRNA expression in lung and breast (BRST) were centered and normalized for each gene, bringing the mean to 0 and the standard deviation to 1. The mean expression of mRNAs correlated with proliferation (on the horizontal axis) was plotted against the mean expression of miRNA markers for tumor/normal distinction (on the vertical axis). Normal samples, poorly differentiated (diff.) tumors and more differentiated tumors are represented by round, triangle and square dots, respectively. Note that the mRNA proliferation signature distinguishes normal samples from tumors, reflecting faster proliferation rates in cancer specimens; however, it does not distinguish between poorly differentiated tumors and more differentiated tumors, even though the miRNA expression levels in the latter two categories are different.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to the discovery and use of improved methods for expression profiling of nucleic acids. As will be discussed in detail below, we have found a simple and flexible method that permits us to rapidly and inexpensively measure gene expression of multiple genes in a single multiplex reaction, ranging from a few genes to 50, 60, 70, 90 or 200 or more genes. Using this method, we have analyzed microRNA and mRNA expression levels, and found these methods are highly efficient and as effective as commercial slide-based microarrays. However, unlike microarrays, the flexibility of the present method permits simple tailoring of the population of genes which can be analyzed in a single reaction. Thus, the present invention is particularly useful for gene expression profiling methods. In addition, using the methods of the invention, we have discovered that microRNAs are downregulated in a wide variety of cancers. Thus, the invention also provides methods for detection of cancer, using microRNA expression profiling.

In one embodiment, the method uses a population of bead sets and measures in solution the expression level of a population of target nucleic acids of interest in a sample. For each individual target nucleic acid of interest, there is a corresponding bead set which comprises a capture probe specific for its target nucleic acid and a unique detectable label, referred to as the bead signal. In this method, a target nucleic acid, such as mRNA in a cell, is first labeled with a detectable signal, referred to as the target signal, before being hybridized with the population of bead sets. Following hybridization in solution of the labeled target nucleic acids with the population of bead sets, the level of both detectable signals is determined for each hybridized bead-target complex. Thus, the bead signal indicates which target nucleic acid is present in the complex, and the level of the target signal indicates the level of expression of that target nucleic acid in the sample. The method can be used to detect tens, or hundreds, or thousands of different target nucleic acids in a single sample.

Accordingly, the invention provides simple, flexible, low-cost, high-throughput methods for simultaneously measuring the expression level of multiple nucleic acids, including mRNAs and microRNAs. In terms of multiplicity, the methods allow the expression level of a few to hundreds, and even thousands, of different target nucleic acids to be measured simultaneously in a single reaction (e.g. 5, 10, 50, 100, 500, or even 1,000 different target nucleic acids). In terms of throughput, the methods allow high numbers of the multiplexed samples to be processed simultaneously, allowing thousands of samples to be rapidly processed. The simplicity of the methods allows the entire procedure to be readily automated. The low cost aspect of the method is reflected for example in a typical unit cost of only several dollars to analyze the expression of 100 nucleic acids in a single sample. As exemplified herein, the performance of the present methods is at least comparable to the current industry-standard oligonucleotide microarrays.

One particularly important advantage of the present method is the high degree of flexibility it provides regarding the population of target nucleic acids to be analyzed. Because the population of bead sets is not fixed, as opposed to the probes on a microarray, the bead population can be readily changed by adding or removing one of the individual bead sets, without altering the other bead sets in the total population. Thus, unlike a slide-based microarray, the population of target nucleic acids to be analyzed can be readily tailored to specific needs, without refabrication of the entire population of bead sets.

The detection methods of the invention can be used in a wide variety of applications as described in detail below, including but not limited to gene expression profiling, screening assays, diagnostic and prognostic assays, for example for gene expression signatures, small molecule or genetic library screening, such as screening cDNA/ORFs, shRNAs, and microRNAs, pharmacogenomics, and the classification of induced biological states.

The invention provides a solution-based method for determining the expression level of a population of target nucleic acids. The method comprises the steps of (a) providing in solution a population of target-specific bead sets, wherein each target-specific bead set is individually detectable and comprises a capture probe which corresponds to an individual target nucleic acid referred to as an individual bead set; (b) hybridizing in solution the population of target-specific bead sets with a population of molecules that can contain a population of detectable target molecules, wherein each target nucleic acid has been transformed into a corresponding detectable target molecule which will specifically bind to its corresponding individual target-specific bead set; and (c) screening in solution for detectable target molecules hybridized to target-specific beads to determine the expression level of the population of target nucleic acids.

In one embodiment, the population of target-specific bead sets comprises at least 5 individual bead sets that can bind with a corresponding set of target nucleic acids. In one embodiment, the population of target-specific beads comprises at least 100 individual bead sets that can bind with a corresponding set of target nucleic acids.

In one embodiment, the population of target nucleic acids is a population of mRNAs. In one embodiment, the population of target nucleic acids is a population of microRNAs.

In one embodiment, each target nucleic acid is an mRNA which has been transformed into a corresponding detectable target molecule. The mRNA is transformed into a corresponding detectable target molecule by a process comprising the steps of (a) reverse transcribing the mRNA target nucleic acid to generate a cDNA; (b) contacting the cDNA with an upstream probe and a downstream probe, wherein the upstream probe comprises a universal upstream sequence and an upstream target-specific sequence, and the downstream probe comprises a universal downstream sequence and a downstream target-specific sequence, such that when the upstream probe and the downstream probe are both hybridized to the cDNA the two probes are capable of being ligated; (c) ligating said cDNA contacted with said upstream and downstream probes to generate ligation complexes; and (d) amplifying said ligation complexes with a pair of universal primers comprising a universal upstream primer and a universal downstream primer. The universal upstream primer is complementary to the universal upstream sequence and the universal downstream primer is complementary to the universal downstream sequence. At least one of the pair of universal primers is detectably labeled. The product of the amplification is detectably labeled. Accordingly, a detectable target molecule is generated which corresponds to the target nucleic acid.

In one embodiment, in the process of transforming the mRNA into a corresponding detectable target molecule, either the upstream probe further comprises an amplicon tag between the universal sequence and the target-specific sequence or the downstream probe further comprises an amplicon tag between the universal sequence and the target-specific sequence. The amplicon tag comprises a nucleic acid sequence that is complementary to the sequence of the capture probe of the bead set.

In one embodiment, each target nucleic acid is a microRNA which has been transformed into a corresponding detectable target molecule. The process of transforming the microRNA into a corresponding detectable target molecule comprises the steps of (a) ligating at least one adaptor to the microRNA, generating an adaptor-microRNA molecule; (b) detectably labeling said adaptor-microRNA molecule. Accordingly, a detectable target molecule is generated which corresponds to the target nucleic acid.

In one embodiment, the adaptor-microRNA is detectably labeled by reverse transcription using the adaptor-microRNA as a template for polymerase chain reaction. In one embodiment, a pair of primers is used in said polymerase chain reaction, and at least one of said primers is detectably labeled.

The present invention further provides a method of screening for the presence of malignancy, infection, cellular disorder, or response to a treatment in a test sample. The method comprises the steps of (a) determining the expression signature of a group of genes in the test sample; and (b) comparing the expression signature between the test sample and a reference sample. A similarity or difference in the expression signature between the test sample and the reference sample is indicative of the presence of malignant cells, infected cells, cellular disorder, or response to a treatment in the test sample. In one embodiment, the solution-based method for determining the expression level of target nucleic acids is used for determination of the expression signature in the test sample and the target nucleic acids are mRNAs. In one embodiment, the expression signature comprises at least 5 genes.

In one embodiment, the reference sample is known to express a predetermined expression signature indicative of the presence of malignancy, infection, or cellular disorder, and the similarity of the expression signature of the test sample to the predetermined expression signature of the reference sample indicates the presence of malignant cells, infected cells, or cellular disorder, in the test sample.

In one embodiment, the reference sample is known to express a predetermined expression signature indicative of a response to treatment, and the similarity of the expression signature of the test sample to the predetermined expression signature of the reference sample indicates the presence of malignant the response to a treatment in the test sample. In one embodiment, the response to treatment is an adverse response to treatment. In one embodiment, the response to treatment is a therapeutic response to treatment.

The invention further provides a method of identifying an expression signature associated with the presence or risk of cancer, infection, cellular disorder, or response to treatment. The method comprises the steps of (a) isolating cells from a group of individuals with said cancer, infection, cellular disorder, or response to treatment, and determining the expression levels of a group of genes; (b) isolating cells from a group of individuals without said cancer, infection, cellular disorder, or response to treatment, and determining the expression levels of said group of genes; and (c) identifying differentially expressed genes from said group of genes which are together indicative of the presence or risk of cancer, infection, cellular disorder, or response to treatment in an individual. Accordingly, an expression signature is identified associated with the presence or risk of cancer, infection, cellular disorder, or response to treatment. In one embodiment, the expression levels of the group of genes is determined using the solution-based method of determining expression level of target nucleic acids.

The invention further provides a method of screening for the presence of malignant cells in a test sample. The method comprises the steps of (a) determining the level of expression of a group of microRNAs in the test sample, and (b) comparing the level of expression of a group of microRNAs between the test sample and a reference sample. In one embodiment, a lower level of expression of the group of microRNAs in the test sample compared to the reference sample is indicative of the test sample containing malignant cells. In one embodiment, a similarity or difference in the level of expression of the group of microRNAs in the test sample compared to the reference sample is indicative of the test sample containing malignant cells. In one embodiment, the microRNAs are transformed into a corresponding detectable target molecule by the process of the present invention. In one embodiment, the determination of the level of microRNA in the sample is determined by the solution-based method of the present invention for determining the expression level of a population of target nucleic acids. In one embodiment, the group of microRNAs comprises at least 5 microRNAs. In one embodiment, the test sample is isolated from an individual at risk of or suspected of having cancer.

The invention further provides a method of screening an individual at risk for cancer. The method comprises the steps of (a) obtaining at least two cell samples from the individual at different times; (b) determining the level of expression of a group of microRNAs in the cell samples, and (c) comparing the level of expression of a group of microRNAs between the cell samples obtained at different times. A lower level of expression of the group of microRNAs in the later obtained cell sample compared to the earlier obtained cell sample is indicative of the individual being at risk for cancer. In one embodiment, the microRNAs are transformed into a corresponding detectable target molecule by the process of the present invention. In one embodiment, the determination of the level of microRNA in the sample is determined by the solution-based method of the present invention for determining the expression level of a population of target nucleic acids.

The invention further provides a method of identifying a microRNA expression signature associated with the presence or risk of cancer, infection, cellular disorder, or response to treatment. The method comprises the steps of (a) isolating cells from a group of individuals with said cancer, infection, cellular disorder, or response to treatment, and determining the expression levels of a group of microRNAs; (b) isolating cells from a group of individuals without said cancer, infection, cellular disorder, or response to treatment, and determining the expression levels of said group of microRNAs; and (c) identifying differentially expressed microRNAs from said group of microRNAs which are together indicative of the presence or risk of cancer, infection, cellular disorder, or response to treatment in an individual. Accordingly, a microRNA expression signature is identified associated with the presence or risk of cancer, infection, cellular disorder, or response to treatment. In one embodiment, the microRNAs are transformed into a corresponding detectable target molecule by the process of the present invention. In one embodiment, the determination of the level of microRNA in the sample is determined by the solution-based method of the present invention for determining the expression level of a population of target nucleic acids.

The invention further provides a method of classifying a tumor sample. The method comprises (a) determining the expression pattern of a group of microRNAs in a tumor sample of unknown tissue origin, generating a tumor sample profile; (b) providing a model of tumor origin microRNA expression patterns based on a dataset of the expression of microRNAs of tumors of known origin; and (c) comparing the tumor sample profile to the model to determine which tumors of known origin the sample most closely resembles. Accordingly, the tissue origin of the tumor sample is classified. In one embodiment, the determination of the level of microRNA in the sample is determined by the solution-based method of the present invention for determining the expression level of a population of target nucleic acids.

The invention further provides a method of classifying a sample from an unknown mammalian species. The method comprises the steps of (a) determining the expression pattern of a group of microRNAs in a sample of an unknown mammalian species, generating a sample profile; (b) providing a model of known mammalian species microRNA expression patterns based on a dataset of the expression of microRNAs of known mammalian species; and (c) comparing the sample profile to the model of known species to determine which known mammalian species the sample profile most closely resembles. Accordingly, the mammalian species of the sample is classified. In one embodiment, the determination of the level of microRNA in the sample is determined by the solution-based method of the present invention for determining the expression level of a population of target nucleic acids.

The invention further provides a method for identifying an active compound or molecule. The method comprises the steps of (a) contacting cells with a plurality of compounds or molecules, (b) determining the expression of a set of marker genes present in the cells using the solution-based method of the present invention for determining the expression level of a population of target nucleic acids, and (c) scoring the expression of the marker genes to identify a cellular phenotype. The presence of a specific cellular phenotype is indicative of an active compound or molecule. In one embodiment, the plurality of chemical compounds or molecules is a set of compounds or molecules selected from the group consisting of small molecule libraries, FDA approved drugs, synthetic chemical libraries, phage display libraries, dosage libraries. In one embodiment, the set of marker genes comprises genes which encode microRNAs and/or messenger RNAs. In one embodiment, the active compound is an anti-cancer drug. In one embodiment, the cellular phenotype is a tumorigenic status of the cell. In one embodiment, the cellular phenotype is a metastatic status of the cell. In one embodiment, the set of marker genes is a cancer versus non-cancer marker gene set. In one embodiment, the set of marker genes is a metastatic versus non-metastatic marker gene set. In one embodiment, he set of marker genes is a radiation resistant versus radiation sensitive marker gene set. In one embodiment, the set of marker genes is a chemotherapy resistant versus chemotherapy sensitive marker gene set. In one embodiment, the active compound is a cellular differentiation factor. In one embodiment, the cellular phenotype is a cellular differentiation status.

The invention further provides a kit for determining in solution the expression level of a population of target nucleic acids. The kit comprises: (a) a population of detectable bead sets, wherein each target-specific bead set is individually detectable and is capable of being coupled to a capture probe which corresponds to an individual target nucleic acid of interest; (b) components for transforming a target nucleic acid of interest into a corresponding detectable target molecule which will specifically bind to its corresponding individual target-specific bead set; and (c) instructions for performing the solution-based method of the present invention for determining the expression level of a population of target nucleic acids. In one embodiment, the population of target nucleic acids comprises mRNAs and the kit further comprises components for performing the method of the present invention for transforming mRNA into a corresponding detectable target molecule. In one embodiment, the population of target nucleic acids comprises microRNAs, and the kit further comprises components for performing the method of the present invention or transforming microRNA into a corresponding detectable target molecule. In one embodiment, the kit further comprises a polymerase and nucleotide bases. In one embodiment, the kit further comprises a plurality of detectable labels. In one embodiment, the kit further comprises capture probes capable of specifically hybridizing to at least 10 different microRNAs, at least 30 different microRNAs, at least 100 different microRNAs, at least 200 different target microRNAs. In one embodiment, the kit further comprises oligonucleotides for use as capture probes or oligonucleotide sequence information to design target specific probes capable of specifically hybridizing to at least 10 different target mRNAs, at least 30 different target mRNAs, at least 100 different target mRNAs, at least 200 different target mRNAs. In one embodiment, the population of target nucleic acids comprises a set of marker genes associated with the presence or risk of cancer, infection, cellular disorder, or response to treatment. In one embodiment, the sample comprises or is suspected of comprising malignant cells.

Samples

The target nucleic acid can be only a minor fraction of a complex mixture such as a biological sample. As used herein, the term “biological sample” refers to any biological material obtained from any source (e.g. human, animal, plant, bacteria, fungi, protist, virus). For use in the invention, the biological sample should contain a nucleic acid molecule. Examples of appropriate biological samples for use in the instant invention include: solid materials (e.g. tissue, cell pellets, biopsies) and biological fluids (e.g. urine, blood, saliva, amniotic fluid, mouth wash).

Nucleic acid molecules can be isolated from a particular biological sample using any of a number of procedures, which are well-known in the art, the particular isolation procedure chosen being appropriate for the particular biological sample.

Solution-Based Method to Determine Expression Levels of Nucleic Acids

The invention provides a solution-based method for highly multiplexed determination of the expression levels of a population of target nucleic acids. The population of target nucleic acids can be a collection of individual target nucleic acids of interest, such as a member of a gene expression signature or just a particular gene of interest. Each individual target nucleic acid of interest is first transformed into a detectable target molecule in a quantitative or semi-quantitative manner, such that the level of each target nucleic acid is reflected by the level of the corresponding detectable target molecule, which is labeled with a detectable signal such as a fluorescent marker. The detectable signal of the target molecule is sometimes referred to as the target molecule signal or simply as the target signal. The method also involves a population of target-specific bead sets, where each target-specific bead set is individually detectable and has a capture probe which corresponds to an individual target nucleic acid. The population of bead sets is hybridized in solution with the population of detectable target molecules to form a hybridized bead-target complex. To determine the expression level of the population of target nucleic acids present, one detects both the target signal and the bead signal for each hybridized bead-target complex, such that the level of the target signal indicates the level of expression of the target nucleic acid, and the bead signal indicates the identity of the target nucleic acid being detected. In one embodiment, the beads can be LUMINEX™ beads, which are polystyrene microspheres that are internally labeled with two spectrally distinct fluorochromes, such that each set of LUMINEX™ beads can be distinguished by its spectral address.

The methods of the invention can be used to detect any population of target nucleic acids of interest, including but not limited to DNAs and RNAs. In one preferred embodiment the target nucleic acids are messenger RNAs (mRNAs). In another preferred embodiment the target nucleic acids are microRNAs (microRNAs).

The present invention provides multiplex detection of target nucleic acids in a sample. As used herein, the phrase multiplex or grammatical equivalents refers to the detection of more than one target nucleic acid of interest within a single reaction. In one embodiment of the invention, multiplex refers to the detection of between 2-10,000 different target nucleic acids in a single reaction. As used herein, multiplex refers to the detection of any range between 2-10,000, e.g., between 5-500 different target nucleic acids in a single reaction, 25-1000 different target nucleic acids, 10-100 different target nucleic acids in a single reaction etc.

The present invention also provides high throughput detection and analysis of target nucleic acids in a sample. As used herein, the phrase “high throughput” refers to the detection or analysis of more than one reaction in a single process, where each reaction is itself a multiplex reaction, detecting more than one target nucleic acid of interest. In one preferred embodiment, 2-10,000 multiplex reactions can be processed simultaneously.

Detectable Bead Sets

The solution-based methods of the invention use detectable target-specific bead sets which comprise a capture probe coupled to a detectable bead, where the capture probe corresponds to an individual target nucleic acid. As used herein, beads, sometimes referred to as microspheres, particles, or grammatical equivalents, are small discrete particles.

Each population of bead sets is a collection of individual bead sets, each of which has a unique detectable label which allows it to be distinguished from the other bead sets within the population of bead sets. In one embodiment, the population comprises at least 5 different individual bead sets. In another embodiment, the population comprises at least 20 different individual bead sets. The population can comprise any number of bead sets as long as there is a unique detectable signal for each bead set. For example, at least 10, 20, 30, 50, 70, 100, 200, 500 or even more different individual bead sets. In a further embodiment, the population comprises at least 1000 different individual bead sets.

Any labels or signals can be used to detect the bead sets as long as they provide unique detectable signals for each bead set within the population of bead sets to be processed in a single reaction. Detectable labels include but are not limited to fluorescent labels and enzymatic labels, as well as magnetic or paramagnetic particles (see, e.g., Dynabeads® (Dynal, Oslo, Norway)). The detectable label may be on the surface of the bead or within the interior of the bead. Detectable labels for use in the invention are described in greater detail below.

The composition of the beads can vary. Suitable materials include any materials used as affinity matrices or supports for chemical and biological molecule syntheses and analyses, including but not limited to: polystyrene, polycarbonate, polypropylene, nylon, glass, dextran, chitin, sand, pumice, agarose, polysaccharides, dendrimers, buckyballs, polyacrylamide, silicon, rubber, and other materials used as supports for solid phase syntheses, affinity separations and purifications, hybridization reactions, immunoassays and other such applications.

Typically the beads have at least one dimension in the 5-10 mm range or smaller. The beads can have any shape and dimensions, but typically have at least one dimension that is 100 mm or less, for example, 50 mm or less, 10 mm or less, 1 mm or less, 100 μm or less, 50 μm or less, and typically have a size that is 10 μm or less such as, 1 μm or less, 100 nm or less, and 10 nm or less. In one embodiment, the beads have at least one dimension between 2-20 μm. Such beads are often, but not necessarily, spherical e.g. elliptical. Such reference, however, does not constrain the geometry of the matrix, which can be any shape, including random shapes, needles, fibers, and elongated. Roughly spherical, particularly microspheres that can be used in the liquid phase, also are contemplated. The beads can include additional components, as long as the additional components do not interfere with the methods and analyses herein.

Commercially available beads which can be used in the methods of the invention include but are not limited to bead-based technologies available from LUMINEX™, Illumina, and Lynx. In one embodiment provides microbeads labeled with different spectral property and/or fluorescent (or colorimetric) intensity. For example, polystyrene microspheres are provided by LUMINEX™ Corp, Austin, Tex. that are internally dyed with two spectrally distinct fluorochromes. Using precise ratios of these fluorochromes, a large number of different fluorescent bead sets (e.g., 100 sets) can be produced. Each set of the beads can be distinguished by its spectral address, a combination of which allows for measurement of a large number of analytes in a single reaction vessel. In this embodiment, the detectable target molecule is labeled with a third fluorochrome. Because each of the different bead sets is uniquely labeled with a distinguishable spectral address, the resulting hybridized bead-target complexes will be distinguishable for each different target nucleic acid, which can be detected by passing the hybridized bead-target complexes through a rapidly flowing fluid stream. In the stream, the beads are interrogated individually as they pass two separate lasers. High speed digital signal processing classifies each of the beads based on its spectral address and quantifies the reaction on the surface. Thousands of beads can interrogated per second, resulting a high speed, high throughput and accurate detection of multiple different target nucleic acids in a single reaction.

In addition to a detectable label, the bead sets also contain a capture probe which corresponds to an individual target nucleic acid. Typically, the capture probes are short unique DNA sequences with uniform hybridization characteristics. Useful capture probes of the invention are described in detail below.

The capture probe can be coupled to the beads using any suitable method which generates a stable linkage between probe and the bead, and permits handling of the bead without compromising the linkage using further methods of the invention. Coupling reactions include but are not limited to the use capture probes modified with a 5′ amine for coupling to carboxylated microsphere or bead.

Methods to Transform a Target mRNA into a Detectable Target Molecule

In one preferred embodiment, the present invention provides methods to detect a population of target nucleic acids, where the target nucleic acids are mRNAs, as illustrated in FIG. 1.

To detect a nucleic acid, for example, mRNAs, the invention provides methods to transform a mRNA into a corresponding detectable target molecule. However, any nucleic acid can be used, e.g., DNA, microRNA, etc. In this example, the mRNA target nucleic acid is first reverse transcribed to generate a cDNA, which is then amplified. During the amplification reaction, a detectable signal is also introduced to create a detectable target molecule, sometimes referred to as a tagged or detectable amplicon. In this process, an upstream probe and a downstream probe are first hybridized to the cDNA. The upstream probe comprises a universal upstream sequence and an upstream target-specific sequence, and the downstream probe comprises a universal downstream sequence and a downstream target-specific sequence, such that when the upstream probe and the downstream probe are both hybridized to the cDNA, the two probes are capable of being ligated, as illustrated in FIG. 1. Next, the upstream and downstream probes hybridized to the cDNA are ligated, to generate a ligation complex. For each mRNA present in the starting sample, a single ligation complex is created. Thus, the number of ligation complexes present is a function of the number of individual mRNA molecules present in the starting sample. Finally, the population of ligation complexes is amplified using a pair of universal primers, a universal upstream primer and a universal downstream primer. The universal upstream primer is complementary to the universal upstream sequence, and the universal downstream primer is complementary to the universal downstream sequence. Typically, the universal upstream sequence and the universal downstream sequence are common between all upstream and downstream probes, respectively, so that within a single multiplex reaction, only two universal primers are required to amplify all of the different target nucleic acids being detected. At least one of the pair of universal primers is detectably labeled, such that the product of the amplification is detectably labeled. Accordingly, this process generates a detectable target molecule which corresponds to the target nucleic acid. Detectable labels are discussed in detail below.

The target-specific sequences of the upstream and the downstream probes comprise polynucleotide sequences that are complementary to a portion of the polynucleotide sequence of the target nucleic acid of interest. Preferably, the target-specific sequences of the present invention are completely complimentary to their corresponding target sequence in the nucleic acid of interest. However, the target-specific sequences used in the present invention can have less than exact complementarity with their target sequences, as long as the upstream and downstream probes hybridized to the target sequence can be ligated by a DNA ligase.

To allow hybridization to the capture probe of the corresponding bead set, a sequence which is complementary to the capture probe must be present in the detectable target molecule. For the detection and analysis of mRNA, this sequence is sometimes referred to as the amplicon tag. The amplicon tag may be a sequence within the target nucleic acid-specific sequence, i.e. part of the upstream or downstream target specific sequences. Alternatively, either the upstream probe or the downstream probe may additionally contain an amplicon tag, which lies between the universal sequence and the target specific sequence of the probe. For example, if the amplicon tag resides within the upstream probe, then it is between the upstream universal sequence and the upstream target specific sequence.

Methods to Transform a microRNA into a Detectable Target Molecule

The present invention also provides methods to detect other nucleic acid, such as a population of microRNAs. The detection of microRNAs represents a significant problem in the art because of their size and sequence similarities. microRNAs are a recently identified class of small non-coding RNAs, which are typically around 21 nucleotides and may differ in sequence by only one or a few nucleotides. At present, hundreds of distinct microRNAs have been identified; however, new microRNAs continue to be described.

Mature microRNAs are excised from a stem-loop precursor that itself can be transcribed as part of a longer primary RNA, sometimes referred to as pri-microRNA. The pri-microRNA is then processed by a nuclear RNAse, cleaving the base of the stem-loop and defining one end of the microRNA. Following export to the cytoplasm, the precursor microRNA is further processed by a second RNAse which cleaves both strands of the RNA, typically about 22 nucleotides from the base of the stem. The two strands of the resulting double-stranded RNA are differentially stable, and the mature microRNA resides on the more stable strand. See Lee, EMBO J. 21:4663-70 (2002); Lee, Nature 425:415-19 (2003); Yi, Genes Dev. 17:17:3011-16 (2003); Lund, Science 303:95-8 (2004); Khvorova, Cell 115:209-16 (2003); and Schwarz, Cell 115:199-208 (2003).

To detect a population of microRNAs, the invention provides methods to transform a microRNA into a corresponding detectable target molecule using essentially the method previously described in Miska et al., Genome Biology 5:R68 (2004). In this method, one first ligates at least one adaptor to the population of microRNAs, generating a population of ligated adaptor-microRNA molecules. These ligated molecules are then detectably labeled, thereby generating a detectable target molecule which corresponds to the specific microRNA. In one embodiment, the adaptor-microRNA is detectably labeled by reverse transcription using the adaptor-microRNA as a template for polymerase chain reaction. At least one of the primers used in said polymerase chain reaction is detectably labeled. Detectable labels are described in detail below.

More particularly, the method involves first size selecting 18-26 nucleotide RNAs from total RNA, for example using denaturing polyacrylamide gel electrophoresis (PAGE). Oligonucleotides are then attached to the 5′ and 3′ ends of the small RNAs to generate ligated small RNAs. The ligated small RNAs are then used as templates for reverse transcription PCR, as previously described for microRNA cloning. See Lee, Science 294:862-4 (2001); Lagos-Quintana, Science 294:853-8 (2001); Lau, Science 294:858-62 (2001). The RT-PCR can include for example 10 cycles of amplification. To detectably label the resulting amplification product, either of the primers used for the RT-PCR reaction can have a detectable label, such as a fluorophore such as Cy3. Preferably, the detectable label is attached to the 5′ end of the primer.

The adaptors of the present invention are comprised of nucleic acid sequences typically not found in the population of microRNAs. Preferably, there is less than 35% identity (homology) between the adaptor sequence and the template, more preferably less than 30% identity, still more preferably less than 25% identity. The sequence analysis programs used to determine homology are run at the default setting.

To specifically identify individual microRNAs, the invention provides a population of bead sets where the capture probes are complementary to the microRNA sequences themselves, rather than the adaptor sequences. Thus, the invention provides in certain embodiments a populations of bead sets which are specific to all known microRNAs. As microRNAs continue to be discovered, the invention allows ready addition of new bead sets corresponding to the newly discovered microRNAs to be added. As discussed in detail below, the invention also provides specific sets of populations of bead sets for the expression profiling of signature microRNAs.

Primers, Probes, and Adaptors

As described above, the probes, primers, and adaptors of the invention comprise include but are not limited to the capture probes of the bead sets, universal primers for amplification of the ligation complexes for nucleic acid detection such as mRNA detection, adaptors for the detection of different nucleic acids such as microRNAs, and amplicon tags for hybridization of the detectable target molecules to the capture probes of the bead sets. The invention also provides additional primers, probes, and adaptors for use in various nucleic acid manipulations. The probes, primers and adaptors are sometimes referred to simply as primers.

The probes, primers, and adaptors used in the methods of the invention can be readily prepared by the skilled artisan using a variety of techniques and procedures. For example, such probes, primers, and adaptors can be synthesized using a DNA or RNA synthesizer. In addition, probes, primers, and adaptors may be obtained from a biological source, such as through a restriction enzyme digestion of isolated DNA. Preferably, the primers are single-stranded.

As used herein, the term “primer” has the conventional meaning associated with it in standard PCR procedures, i.e., an oligonucleotide that can hybridize to a polynucleotide template and act as a point of initiation for the synthesis of a primer extension product that is complementary to the template strand.

Preferably, the primers of the present invention have exact complementarity with its target sequence. However, primers used in the present invention can have less than exact complementarity with their target sequence as long as the primer can hybridize sufficiently with the target sequence so as to function as described; for example to be extendible by a DNA polymerase or for hybridization with the capture probe of the bead set.

For use in a given multiplex reaction, the universal primer sequences are typically analyzed as a group to evaluate the potential for fortuitous dimer formation between different primers. This evaluation may be achieved using commercially available computer programs for sequence analysis, such as Gene Runner, Hastings Software Inc. Other variables, such as the preferred concentrations of Mg⁺², dNTPs, polymerase, and primers, are optimized using methods well-known in the art (Edwards et al., PCR Methods and Applications 3:565 (1994)).

Detectable Labels

Any labels or signals which allow detection of the bead set and the detectable target molecules can be used in the methods of the invention. Such detectable labels are well known in the art.

According to the invention, there is a target-specific bead set which corresponds to each target nucleic acid of interest. For each bead set there is a detectable signal, and for the corresponding target nucleic acid there is a distinct detectable signal. Thus, detection of an individual target nucleic interest requires two distinguishable detectable signals.

The detectable labels of the invention may be added to the target nucleic acid and/or the bead sets using various methods. The detectable label may be covalently conjugated with the nucleic acid or non-covalently attached to the nucleic through sequence-specific or non-sequence-specific binding. Examples of the detectable labels include, but are not limited to biotin, digoxigenin, fluorescent molecule (e.g., fluorescin and rhodamine), chemiluminescent moiety (e.g., LUMINOL™), coenzyme, enzyme substrate, radio isotopes, a particle such as latex or carbon particle, nucleic acid-binding protein, polynucleotide that specifically hybridizes with either the target or reference nucleic acid strand. Detection of the presence of the label can be achieved by observation or measurement of signals emitted from the label. The production of the signal may be facilitated by binding of the label to its counter-part molecule, which triggers a reaction directly or indirectly. For example, the target nucleic acid may be labeled with biotin; upon binding of streptavidin-HRP (horse radish peroxidase) and addition of the substrate for HRP (e.g., ABTS), the presence of the biotin-labeled target molecule can be detected by observing or measuring color changes in the mixture.

In certain preferred embodiments, the labels are fluorescent and the hybridized bead-target complexes are detected using fluorescence polarization machine, also referred to as a flow cytometer. Fluorescent dyes with diverse spectral properties (e.g., as supplied by MOLECULAR PROBES™, Eugene, Oreg.) may be used to simultaneously detect multiple detectable target molecules. In this assay, each target molecules may be labeled with a fluorescent dye having different spectral property than that for another target molecule. In another preferred embodiment, the detectable target molecule is labeled with a biotin, and the final hybridized bead-target complexes are further reacted with a signal such as streptavidin-phycoerythrin.

Target Nucleic Acids

In the present invention, a target nucleic acid refers to a sequence of nucleotides to be studied either for the presence of a difference from a reference sequence or for the determination of its presence or absence. The target nucleic acid sequence may be double stranded or single stranded and from a natural or synthetic source. When the target nucleic acid sequence is single stranded, a nucleic acid duplex comprising the single stranded target nucleic acid sequence may be produced by primer-extension and/or amplification.

The present invention is preferably used with at least 5 targets in a single reaction, more preferably at least 10 targets, still more preferably with at least 14 targets, even more preferably with at least 20 targets, yet more preferably with at least 30 targets, still more preferably with at least 50 targets, and even more preferably with at least 100 targets in a single reaction, although one can target any number from 5-1000 as long as a uniquely detectable signal is used. Multiplex detection as used herein refers to the simultaneous detection of multiple nucleic acid targets in a single reaction mixture.

High-throughput denotes the ability to simultaneously process and screen a large number of individual reaction mixtures such as multiplexed nucleic acid samples (e.g. in excess of 100 RNAs) in a rapid and economical manner, as well as to simultaneously screen large numbers of different target nucleic acids within a single multiplexed nucleic acid sample.

Any nucleic acid sample of interest may be used in practicing the present invention, including without limitation eukaryotic, prokaryotic and viral DNA or RNA. In a preferred embodiment, the target nucleic acids represents a sample of total RNA, including mRNA and microRNA, isolated from an individual. This DNA may be obtained from any cell source or body fluid. Non-limiting examples of cell sources available in clinical practice include blood cells, buccal cells, cervicovaginal cells, epithelial cells from urine, fetal cells, or any cells present in tissue obtained by biopsy. Body fluids include blood, urine, cerebrospinal fluid, semen and tissue exudates at the site of infection or inflammation. Nucleic acid such as RNA is extracted from the cell source or body fluid using any of the numerous methods that are standard in the art. It will be understood that the particular method used to extract the nucleic acid will depend on the nature of the source and the type of nucleic acid to be extracted.

The present method can be used with polynucleotides comprising either full-length RNA or DNA, or their fragments. The RNA or DNA can be either double-stranded or single-stranded, and can be in a purified or unpurified form. Preferably, the polynucleotides are comprised of RNA. In certain embodiments, the present invention can be used with full-size cDNA polynucleotide sequences, such as can be obtained by reverse transcription of RNA. The DNA fragments used in the present invention can be obtained by digestion of cDNA with restriction endonucleases, or by amplification of cDNA fractions from cDNA using arbitrary or sequence-specific PCR primers. The nucleic acid can be obtained from a variety of sources, including both natural and synthetic sources. The nucleic acid can be from any natural source including viruses, bacteria, yeast, plants, insects and animals.

Certain embodiments of the invention provide amplification of a nucleic acid using polymerase chain reaction (PCR). “Amplification” of DNA as used herein denotes the use of polymerase chain reaction (PCR) to increase the concentration of a particular DNA sequence within a mixture of DNA sequences. In practicing the present invention, a nucleic acid sample is contacted with pairs of oligonucleotide primers under conditions suitable for polymerase chain reaction. Conditions for performing PCR are well known in the art. Standard PCR reaction conditions may be used, e.g., 1.5 mM MgCl.sub.2, 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 200 μM deoxynucleotide triphosphates (dNTPs), and 25-100 U/ml Taq polymerase (PERKIN-ELMER™, Norwalk, Conn.). The concentration of each primer in the reaction mixture can range from about 0.05 to about 4 μM. Each potential primer can be evaluated by performing single PCR reactions using each primer pair (e.g. a universal upstream primer and a universal downstream primer) individually. Similarly, each primer pair can be evaluated independently to confirm that all primer pairs to be included in a single multiplex PCR reaction generate a product of the expected size. As the number of targets in a single reaction increases, certain targets may not be amplified as efficiently as other targets. The concentration of the primers for such underrepresented targets may be increased to increase their yield. For example, when multiplying 15 or more targets; more preferably, when multiplying 30 or more targets.

Multiplex PCR reactions are typically carried out using manual or automatic thermal cycling. Any commercially available thermal cycler may be used, such as, e.g., PERKIN-ELMER™ 9600 cycler.

A variety of DNA polymerases can be used during PCR with the present invention. Preferably, the polymerase is a thermostable DNA polymerase such as may be obtained from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermus flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu). Many of these polymerases may be isolated from the bacterium itself or obtained commercially. Polymerases to be used with the present invention can also be obtained from cells which express high levels of the cloned genes encoding the polymerase. Preferably, a combination of several thermostable polymerases can be used.

The PCR conditions used to amplify the targets are standard PCR conditions which are well known in the art. Typical conditions use 35-40 cycles, with each cycle comprising a denaturing step (e.g. 10 seconds at 94° C.), an annealing step (e.g. 15 sec at 68° C.), and an extension step (e.g. 1 minute at 72° C.). As the number of targets in a single reaction increases, the length of the extension time may be increased. For example, when amplifying 30 or more targets, the extension time may be three times as longer than when amplifying 10-15 targets (e.g. 3 minutes instead of 1 minute).

In addition to the detection methods specific to the present invention, the reaction products can be analyzed using any of several methods that are well-known in the art, for example to confirm isolated steps of the methods. For example, agarose gel electrophoresis can be used to rapidly resolve and identify each of the amplified sequences. In a multiplex reaction, different amplified sequences are preferably of distinct sizes and thus can be resolved in a single gel. In one embodiment, the reaction mixture is treated with one or more restriction endonucleases prior to electrophoresis. Alternative methods of product analysis include without limitation dot-blot hybridization with allele-specific oligonucleotides and SSCP.

Applications

The methods of the invention can be used in any application or method in which it is desirable to measure or detect the presence of a population of target nucleic acids, such as for gene expression profiling or microRNAs profiling. While several preferred applications are described in detail here, the invention is in no way limited to these embodiments. Other applications would become apparent to one skilled in the art having the benefit of this disclosure.

As described in detail below, the invention can be used in methods for gene expression profiling assays such as, diagnostic and prognostic assays, for example for gene expression signatures, molecule or genetic library screening, such as screening cDNA/ORFs, shRNAs, and microRNAs, pharmacogenomics, and the classification of induced biological states.

Expression Profiling Applications

The methods of the invention are useful for a variety of gene expression profiling applications. More particularly, the invention encompasses methods for high-throughput genetic screening. The method allows the rapid and simultaneous detection of multiple defined target nucleic acids such as mRNA or microRNA sequences in nucleic samples obtained from a multiplicity of individuals. It can be carried out by simultaneously amplifying many different target sequences from a large number of desired samples, such as patient nucleic acid samples, using the methods described above.

In general, as used herein, an expression signature is a set of genes, where the expression level of the individual genes differs between a first physiological state or condition relative to their expression level in a second physiological state or condition, i.e. state A and state B. For example, between cancerous cells and non-cancerous cells, or cells infected with a pathogen and uninfected cells, or cells in different states of development.

The terms “differentially expressed gene,” “differential gene express” and their synonyms, which are used interchangeably, refer to a gene whose expression is activated to a higher or lower level in one physiological state relative to a second physiological subject suffering from a disease, such as cancer, relative to its expression in a normal or control subject. As used herein, “gene” specifically includes nucleic acids which do not encode proteins, such as microRNAs. The terms also include genes whose expression is activated to a higher or lower level at different states of the same disease. A differentially expressed gene may be either activated or inhibited at the nucleic acid level or protein level, or may be subject to alternative splicing to result in a different polypeptide product. Such differences may be evidenced by a change in mRNA levels or microRNA levels, surface expression, secretion or other partitioning of a polypeptide, for example. Differential gene expression may include a comparison of expression between two or more genes or their gene products, or a comparison of the ratios of the expression between two or more genes or their gene products, or even a comparison of two differently processed products of the same gene, which differ between normal subjects and subjects suffering from a disease, specifically cancer, or between various stages of the same disease. Differential expression includes both quantitative, as well as qualitative, differences in the temporal or cellular expression pattern in a gene or its expression products among, for example, normal and diseased cells, or among cells which have undergone different disease events or disease stages. Differential gene expression is considered to be present when there is at least an about two-fold, preferably at least about four-fold, more preferably at least about six-fold, more preferably at least about ten-fold difference between the expression of a given gene between two different physiological states, such as in various stages of disease development in a diseased individual.

An expression signature is sometimes referred to herein as a set of marker genes. An expression signature, or set of marker genes, is a minimum number of genes that is capable of identifying a phenotypic state of a cell. A set of marker genes that is representative of a cellular phenotype is one which includes a minimum number of genes that identify markers to demonstrate that a cell has a particular phenotype. In general, two discrete cell populations in different physiological states having the desired phenotypes may be examined by the methods of the invention. The minimum number of genes in a set of marker genes will depend on the particular phenotype being examined. In some embodiments the minimum number of genes is 2 or, more preferably, 5 genes. In other embodiments, the minimum number of genes is 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1000 genes.

Screening for Expression Signatures

One embodiment of the invention provides highly practical, i.e. low cost, high throughput, and highly flexible routine mRNA expression analysis, for example for clinical testing. The invention provides methods to analyze the expression signature for a cellular phenotype of interest by determining the expression level of a set of marker genes in a test sample. A “phenotype” as used herein refers to a physiological state of a cell under a specific set of conditions, including but not limited to malignancy, infection or a cellular disorder.

In general, analysis of an expression signature involves first determining the expression profile of a gene group, also known as the expression signature, in the test sample, and comparing the expression profile between the test sample and a corresponding control sample, where a difference in the expression profile between the test sample and the control sample is indicative of the test sample expressing the physiological state or cellular phenotype associated with the signature profile. There can be a range of differences in gene expression in the expression profile between the control sample and the profile of interest. Preferably, there are differences from the control profile in at least 25% of the genes being looked at. This can range from a sample showing a 25% change to 100% change from the control sample pattern to the condition of interest and all points in between, at least 30%, at least 40%, at least 50%, at least 75%, at least 90%.

The methods of the invention can be used to analyze any expression signature for a cellular phenotype of interest. The identification of expression signatures is the subject of intense study. The invention contemplates the analysis of any expression signature of interest and is in no way limited to the specific embodiments described herein.

In one embodiment, the present invention provides methods to measure gene expression signatures in a sample, where the expression signature is indicative of a malignancy. For example, van de Vivjer et al. New Engl. J. Med. 347: 1999-2009 (2002) described a 70 member expression signature associated with breast cancer malignancy or metastasis, and is a predictor of survival. U.S. Patent Application Publication No. 2004/0018527 discloses a group of 91 genes associated with docetaxel chemosensitivity in breast cancer. Additional breast cancer expression signatures are described in detail in U.S. Patent Application Publication No. 2004/0058340 as well as Abba et al., BMC Genomics 6:37 (2005). Glas et al. (2005) described an 81 member expression signature associated with follicular lymphoma, particularly the aggressiveness of the lymphoma. Stegmaier et al. (2004) described a 5 member expression signature which was used in a cell-based small molecule screen for agents inducing the differentiation of human leukemia cells. U.S. Patent Application Publication No. 2004/0009523 discloses 14 genes associated with a diagnosis of multiple myeloma, as well as four subgroups of 24 genes associated with a prognosis of multiple myeloma. U.S. Patent Application Publication No. 2005/0089895 discloses 26 genes associated with the likelihood of recurrence in hepatocellular carcinoma. O'Donnell et al., 2005, Oncogene 24:1244-51, described a group of 116 genes associated with squamous cell carcinoma of the oral cavity. Beer et al. 2002, Nat Med 8:816-824 discloses 50 gene risk index associated with lung adenocarcinoma survival. Classification of human lung cancer by gene expression profiling has been described in several recent publications (M. Garber, PNAS, 98(24): 13784-13789 (2001); A. Bhattacharjee, PNAS, 98(24):13790-13795 (2001). Ramaswamy et al., 2002, Nat Gen 33:49-54 discloses 128 genes whose relative expression levels distinguish between primary and metastatic tumors. Glinsky et al., 2005, J. Clin. Invest. 115:1503-21, discloses 11 genes associated with highly aggressive disease outcomes for several different cancers.

Other disease conditions have also been found to be associated with expression signatures. For example, U.S. Patent Application Publication No. 20040220125 discloses 40 cardioprotective genes, which are useful as a means to diagnose cardiopathology. Baechler et al. 2003, PNAS 100:2610-15 disclose a group of 161 genes associated with severe lupus; see also U.S. Patent Application Publication No. 2004/0033498.

Other cellular states for which expression signatures have been reported include apoptosis, for which a set of 35 regulator genes has been reported (Eldering et al., Nuc. Acid Res. 31:e153 (2003), as well as inflammation, which was associated with a group of 30 genes (Id.).

The present invention also provides methods for diagnosis of infection by gene expression profiling using the methods of the invention. In one embodiment, the expression signature is comprised of cellular host genes whose expression is altered in the presence of an infectious agent. For example, U.S. Patent Application Publication No. 20040038201 discloses expression signatures of cellular host genes associated with infection with a variety of infectious agents, including E. coli, the enterohemorrhagic pathogen E. coli 0157:H7, Salmonella spp. Staphylococcus aureus, Listeria monocytogenes, M. tuberculosis, and M. bovis bacilli Calmette-Gurin (BCG).

In another embodiment, the expression signature is comprised of genes of the infectious agent. The expression signature can also comprise a combination of host and infectious agent genes.

Another preferred embodiment of the invention provides methods for screening for the presence of an infection in a sample by detecting the presence of multiple genes associated with the infectious agent. Viruses, bacteria, fungi and other infectious organisms contain distinct nucleic acid sequences, which are different from the sequences contained in the host cell. Detecting or quantifying nucleic acid sequences that are specific to the infectious organism is important for diagnosing or monitoring infection. Examples of disease causing viruses that infect humans and animals and which may be detected by the disclosed processes include but are not limited to: Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, See Ratner, L. et al., Nature, Vol. 313, Pp. 227-284 (1985); Wain Hobson, S. et al, Cell, Vol. 40: Pp. 9-17 (1985)); HIV-2 (See Guyader et al., Nature, Vol. 328, Pp. 662-669 (1987); European Patent Publication No. 0 269 520; Chakraborti et al., Nature, Vol. 328, Pp. 543-547 (1987); and European Patent Application No. 0 655 501); and other isolates, such as HIV-LP (International Publication No. WO 94/00562 entitled “A Novel Human Immunodeficiency Virus”; Picornaviridae (e.g., polio viruses, hepatitis A virus, (Gust, I. D., et al., Intervirology, Vol. 20, Pp. 1-7 (1983); entero viruses, human coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses); Birnaviridae, Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes viruses); Poxyiridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class 1=internally transmitted; class 2=parenterally transmitted (i.e., Hepatitis C); Norwalk and related viruses, and astroviruses).

Examples of infectious bacteria include but are not limited to: Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g. M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus antracis, corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, and Actinomyces israelli.

Examples of parasitic protozoan infections include but are not limited to: Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium falciparum, Toxoplasma gondii, Pneumocystis carinii, Trypanosoma cruzi, Trypanasoma brucei gambiense, Trypanasoma brucei rhodesiense, Leishmania species, including Leishmania donovani, Leishmania mexicana, Naegleria, Acanthamoeba, Trichomonas vaginalis, Cryptosporidium species, Isospora species, Balantidium coli, Giardia lamblia, Entamoeba histolytica, and Dientamoeba fragilis. See generally, Robbins et al, Pathologic Basis of Disease (Saunders, 1984) 273-75, 360-83.

microRNA Expression Profiles

We have also found that one can screen for the presence of malignant cells in a test sample by determining the level of expression of total microRNAs in a test sample; and comparing the levels of expression of microRNAs of the test sample and a control sample. A lower level of expression of microRNAs in the test sample compared to the control sample is indicative of the test sample containing malignant cells. One can use any screening method including the solution base method described herein, or other known methods such as micorarrays for microRNAs, such as that described in Miska et al., 2004.

Another embodiment of the invention provides methods of screening an individual at risk for cancer by obtaining at least two cell samples from the individual at different times; and comparing the level of expression of microRNAs in the cell samples, where a lower level of expression of microRNAs in the later obtained cell sample compared to the earlier obtained cell sample indicates that the individual is at risk for cancer.

In one preferred embodiment, the methods of the present invention are useful for characterizing poorly differentiated tumors. As exemplified herein, microRNA expression distinguishes tumors from normal tissues, even for poorly differentiated tumors. As shown in FIG. 9, the majority of microRNAs analyzed were expressed in lower levels in tumors compared to normal tissues, irrespective of cell type.

The methods of detecting microRNAs are particularly useful for detecting tumors of histologically uncertain cellular origin, which account for 2-4% of all cancer diagnoses. In this embodiment, the expression profile of microRNAs in a tumor of uncertain cellular origin is compared to a set of microRNA expression profiles for a set of tumors of known origin, allowing classification of the test samples to be assessed based on the comparison.

In another embodiment, the level of expression for a specific group of microRNAs, sometimes referred to a profile group of microRNAs, is determined, where lower expression of said profile group of microRNAs is associated with risk for a particular type of cancer. In particular, microRNAs can be used to classify acute lymphoblastic leukemias into the following subclassifications: t(9;22) BCR/ABL ALLs; t(12;21) TEL/AML1 ALLs; and T-cell ALLs.

Identification of Novel Expression Signatures

We have also discovered methods for identifying an expression profile of a gene group associated with risk of a cellular disorder. It can be any type of nucleic acid that is viewed. In certain embodiments, the genes encode mRNAs. In other preferred embodiments, the genes encode microRNAs.

In one embodiment, the methods involve the establishment of two or more sets of gene expression profiles. The gene expression profiles are utilized to develop marker gene sets which identify a phenotype. Thus, the methods of the invention involve the identification of a cell signature which is useful for identifying a phenotype of a cell.

As used herein, a control gene or set of control genes is selected that are common between the two physiological states in similar or equivalent degrees of gene expression. Additionally, a common housekeeping gene(s) may be used as an “internal” reference or control to normalize the readout for relative differences in cell populations in the screening assay. One example of a common gene useful in the invention is glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (M33197). The expression level of the marker genes will define the phenotypic state when taken in ratio to the common gene(s). Hence, quantitation of the expression levels for 2 or more marker genes will be adequate to identify a new phenotypic state.

In this method, one isolates cells from a group of individuals with a cancer, infection, or cellular disorder, and determining the expression level of multiple genes; isolating cells from a group of individuals without said cancer, infection, or cellular disorder, and determining the expression level of said multiple genes; and identifying differential gene expression patterns that are statistically significant; and applying linear regression analysis to identify an expression profile of a gene group that is indicative of an individual having risk of said cancer, infection, or cellular disorder. One can use any screening technique to identify the expression profile. The method described herein is particularly useful because of the flexibility it provides in selecting beads that suit a specific profile.

Small Molecule Screening Methods

The present invention also provides methods to screen a library to identify molecules that change the profile of a cell to result in a desired result. The methods of multiplex target nucleic acid detection are particularly useful in methods for drug screening, such as those disclosed in U.S. Published Patent Application No. 2004/0009495, which is hereby incorporated herein in its entirety.

In this method, the effect of a molecule such as a small molecule protein, etc. on the expression profile signature is used to identify small molecules of interest. For example, one can screen for molecules which alter an expression signature associated with a biological state, such as cancer, such that the expression signature of a sample exposed to the small molecule is altered to more closely resemble the healthy state, i.e. a non-cancerous state. One would look for molecules that change the profile of at least 25% of the genes in the profiling to a profile of the healthy cell. In. other embodiments, one looks for molecules or groups of molecules that result in a change of the expression profile of at least 30$, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90% until one gets virtual identity with the desired state.

In another embodiment, one can also screen from molecules that cause an undesired condition by looking at how an expression profile is changes from the desired profile to an undesired profile. The present methods can also be used to monitor when a patient should get therapy, what therapy and the effect of that therapy. For example, in pharmacogenomics applications and methods, including the use of gene expression signatures to predict response to therapy. Such applications can be deployed on this platform providing a practical (i.e. low cost, high throughput) mRNA expression based tool to inform treatment decisions or enrollment in clinical trials.

The screening methods may be used for identifying therapeutic agents or validating the efficacy of agents. Agents of either known or unknown identity can be analyzed for their effects on gene expression in cells using methods such as those described herein. Briefly, purified populations of cells are exposed to the plurality of chemical compounds, preferably in an in vitro culture high throughput setting, and optionally after set periods of time, the entire cell population or a fraction thereof is removed and mRNA is harvested therefrom. Any target nucleic acids, such as mRNAs or microRNAs, are then analyzed for expression of marker genes using methods such as those described herein. Hybridization or other expression level readouts may be then compared to the marker gene data. These methods can be used for identifying novel agents, as well as confirming the identity of agents that are suspected of playing a role in regulation of cellular phenotype.

The methods of the invention allows for subjects to be screened and potentially characterized according to their ability to respond to a plurality of drugs. For instance, cells of a subject, e.g., cancer cells, may be removed and exposed to a plurality of putative therapeutic compounds, e.g., anti-cancer drugs, in a high throughput manner. The nucleic acids of the cells may then be screened using the methods described herein to determine whether marker genes indicative of a particular phenotype are expressed in the cells. These techniques can be used to optimize therapies for a particular subject. For instance, a particular anti-cancer therapy may be more effective against a particular cancer cell from a subject. This could be determined by analyzing the genes expressed in response to the plurality of compounds. Likewise a therapeutic agent with minimal side effects may be identified by comparing the genes expressed in the different cells with a marker gene set that is indicative of a phenotype not associated with a particular side effect. Additionally, this type of analysis can be used to identify subjects for less aggressive, more aggressive, and generally more tailored therapy to treat a disorder.

The methods are also useful for determining the effect of multiple drugs or groups of drugs on a cellular phenotype. For instance it is possible to perform combined chemical genomic screens to identify a synergistic or other combined effect arising from combinations of drugs. One set of drugs that induces a first set of marker genes indicative of a phenotype, while another drug induces an second set of marker genes. When the two sets of drugs are combined they may act to achieve a collective phenotypic change, exemplified by a third set of marker genes. Additionally the methods could be used to assess complex multidrug effects on cell types. For instance, some drugs when used in combination produce a combined toxic effect. It is possible to perform the screen to identify marker genes associated with the toxic phenotype. Existing compounds could be screened for there ability to “trip” the signal signature of toxic effect, by monitoring the marker genes associated with the toxic phenotype.

The methods may also be used to enhance therapeutic strategies. For instance, oncolytic therapy involves the use of viruses to selectively lyse cancer cells. A set of marker genes which identify a gene expression signature favorable to selective viral infection can be identified. Using this set of marker genes, drugs can be found which favor or enable selective viral infectivity in order to enhance the therapeutic benefit.

Thus, the methods of the invention are useful for screening multiple compounds. For instance, the methods are useful for screening libraries of molecules, FDA approved drugs, and any other sets of compounds. Preferably the methods are used to screen at least 20 or 30 compounds, and more preferably, at least 50 compounds. In some embodiments, the methods are used to screen more than 96, 384, or 1536 compounds at a time.

In one embodiment, the methods of the invention are useful for screening FDA approved drugs. An FDA approved drug is any drug which has been approved for use in humans by the FDA for any purpose. This is a particularly useful class of compounds to screen because it represents a set of compounds which are believed to be safe and therapeutic for at least one purpose. Thus, there is a high likelihood that these drugs will at least be safe and possibly be useful for other purposes. FDA approved drugs are also readily commercially available from a variety of sources.

A “library of molecules” as used herein is a series of molecules displayed such that the compounds can be identified in a screening assay. The library may be composed of molecules having common structural features which differ in the number or type of group attached to the main structure or may be completely random. Libraries are meant to include but are not limited to, for example, phage display libraries, peptides-on-plasmids libraries, polysome libraries, aptamer libraries, synthetic peptide libraries, synthetic small molecule libraries and chemical libraries. Methods for preparing libraries of molecules are well known in the art and many libraries are commercially available. Libraries of interest include synthetic organic combinatorial libraries. Libraries, such as, synthetic small molecule libraries and chemical libraries. The libraries can also comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more functional groups. Libraries of interest also include peptide libraries, randomized oligonucleotide libraries, and the like. Degenerate peptide libraries can be readily prepared in solution, in immobilized form as bacterial flagella peptide display libraries or as phage display libraries. Peptide ligands can be selected from combinatorial libraries of peptides containing at least one amino acid. Libraries can be synthesized of peptoids and non-peptide synthetic moieties. Such libraries can further be synthesized which contain non-peptide synthetic moieties which are less subject to enzymatic degradation compared to their naturally-occurring counterparts.

Small molecule combinatorial libraries may also be generated. A combinatorial library of small organic compounds is a collection of closely related analogs that differ from each other in one or more points of diversity and are synthesized by organic techniques using multi-step processes. Combinatorial libraries include a vast number of small organic compounds. One type of combinatorial library is prepared by means of parallel synthesis methods to produce a compound array. A “compound array” as used herein is a collection of compounds identifiable by their spatial addresses in Cartesian coordinates and arranged such that each compound has a common molecular core and one or more variable structural diversity elements. The compounds in such a compound array are produced in parallel in separate reaction vessels, with each compound identified and tracked by its spatial address. Examples of parallel synthesis mixtures and parallel synthesis methods are provided in U.S. Pat. No. 5,712,171 issued Jan. 27, 1998.

One type of library, which is known as a phage display library, includes filamentous bacteriophage which present a library of peptides or proteins on their surface. Phage display libraries can be particularly effective in identifying compounds which induce a desired effect in cells. Briefly, one prepares a phage library (using e.g. m13, fd, lambda or T7 phage), displaying inserts from 4 to about 80 amino acid residues using conventional procedures. The inserts may represent, for example, a completely degenerate or biased array. DNA sequence analysis can be conducted to identify the sequences of the expressed polypeptides. The minimal linear peptide or amino acid sequence that have the desired effect on the cells can be determined. One can repeat the procedure using a biased library containing inserts containing part or all of the minimal linear portion plus one or more additional degenerate residues upstream or downstream thereof.

For certain embodiments of this invention, e.g., where phage display libraries are employed, a preferred vector is filamentous phage, though other vectors can be used. Vectors are meant to include, e.g., phage, viruses, plasmids, cosmids, or any other suitable vector known to those skilled in the art. The vector has a gene, native or foreign, the product of which is able to tolerate insertion of a foreign peptide. By gene is meant an intact gene or fragment thereof. Filamentous phage are single-stranded DNA phage having coat proteins. Preferably, the gene that the foreign nucleic acid molecule is inserted into is a coat protein gene of the filamentous phage. Examples of coat proteins are gene III or gene VIII coat proteins. Insertion of a foreign nucleic acid molecule or DNA into a coat protein gene results in the display of a foreign peptide on the surface of the phage. Examples of filamentous phage vectors which can be used in the libraries are fUSE vectors, e.g., fUSE1 fUSE2, fUSE3 and fUSE5, in which the insertion is just downstream of the pill signal peptide. Smith and Scott, Methods in Enzymology 217:228-257 (1993).

By recombinant vector it is meant a vector having a nucleic acid sequence which is not normally present in the vector. The foreign nucleic acid molecule or DNA is inserted into a gene present on the vector. Insertion of a foreign nucleic acid into a phage gene is meant to include insertion within the gene or immediately 5′ or 3′ to, respectively, the beginning or end of the gene, such that when expressed, a fusion gene product is made. The foreign nucleic acid molecule that is inserted includes, e.g., a synthetic nucleic acid molecule or a fragment of another nucleic acid molecule. The nucleic acid molecule encodes a displayed peptide sequence. A displayed peptide sequence is a peptide sequence that is on the surface of, e.g. a phage or virus, a cell, a spore, or an expressed gene product.

In certain embodiments, the libraries may have at least one constraint imposed upon their members. A constraint includes, e.g., a positive or negative charge, hydrophobicity, hydrophilicity, a cleavable bond and the necessary residues surrounding that bond, and combinations thereof. In certain embodiments, more than one constraint is present in each of the broader sequences of the library.

In addition to the basic libraries, the methods can also be used to screen combinations of drugs. Thus, more than one type of drug can be contacted with each cell.

In other aspects of the invention, the cells do not necessarily need to be contacted with any compounds. The cells may be analyzed for phenotypic status based on environmental condition, such as in vivo or in vitro conditions. It is possible to analyze the differentiation state or tumorigenic state of a cell using the marker gene sets or metagenes of the invention. Thus, a cell may be subjected to conditions in vitro or in vivo and then analyzed for differentiation status.

Additionally, it is possible to screen sets of compounds to identify particular dosages effective at producing a phenotypic state in a cell. For instance, one or more drugs could be contacted with the cells at a variety of dosages over a large range. When the level of marker genes expressed in each of the cells is assessed, it will be possible to identify an optimum dosage for producing a particular phenotypic state of the cell. Additionally, if some markers are associated with the production of undesirable side effects, such as production of cytotoxic factors, then an optimum drug, combination of drug or dosage of drug can be identified using the methods of the invention.

The methods of the invention are useful for assaying the effect of compounds on cells or for analyzing the phenotypic status of a cell. The methods may be used on any type of cell known in the art. For instance the cell may be a cultured cell line or a cell isolated from a subject (i.e. in vivo cell population). The cell may have any phenotypic property, status or trait. For instance, the cell may be a normal cell, a cancer cell, a genetically altered cell, etc.

Cancers include, but are not limited to, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer; intra-epithelial neoplasm; kidney cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small cell and non-small cell); lymphoma including Hodgkin's and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; renal cancer; cancer of the respiratory system; sarcoma; skin cancer; stomach cancer; testicular cancer; thyroid cancer; uterine cancer; cancer of the urinary system, as well as other carcinomas and sarcomas. Some cancer cells are metastatic cancer cells.

“Normal cells” as used herein refers any cell, including but not limited to mammalian, bacterial, plant cells, that is a non-cancer cell, non-diseased, or a non-genetically engineered cell. Mammalian cells include but are not limited to mesenchymal, parenchymal, neuronal, endothelial, and epithelial cells.

A “genetically altered cell” as used herein refers to a cell which has been transformed with an exogenous nucleic acid.

Kits

The present invention further concerns kits which contain, in separate packaging or compartments, the reagents such as adaptors and primers required for practicing the detection methods of the invention. Such kits typically include at least a population of detectable bead sets and preferably several different primers to generate a population of detectably labeled target molecules for detection. Such kits may optionally include the reagents required for performing ligation reactions, such as DNA or RNA ligases, PCR reactions, such as DNA polymerase, DNA polymerase cofactors, and deoxyribonucleotide-5′-triphosphates. Optionally, the kit may also include various polynucleotide molecules, restriction endonucleases, reverse transcriptases, terminal transferases, various buffers and reagents. Optimal amounts of reagents to be used in a given reaction can be readily determined by the skilled artisan having the benefit of the current disclosure.

The kits may also include reagents necessary for performing positive and negative control reactions. Preferably the kits include several target nucleic acids, in separate vials or tubes, or preferably, a set of combined standards comprising at least two different standards in the same vial or tube with known amount of dried standard nucleic acid(s) with instructions to dilute the sample in a suitable buffer, such as PBS, to a known concentration for use in the quantification reaction. Alternatively, the standard is pre-diluted at a known concentration in a suitable buffer, such as PBS. Suitable buffer can be either suitable for both for storing nucleic acids and for, e.g., PCR or direct enhancement reactions to enhance the difference between the standard and a corresponding target nucleic acid as described above, or the buffer is just for storing the sample and a separate dilution buffer is provided which is more suitable for the consequent PCR, enhancement and quantification reactions. In a preferred embodiment, all the standard nucleic acids are combined in one tube or vial in a buffer, so that only one standard mix can be added to a nucleic acid sample containing the target nucleic acid.

The kit also preferably comprises a manual explaining the reaction conditions and the measurement of the amount of target nucleic acid(s) using the standard nucleic acid(s) or a mixture of them and gives detailed concentrations of all the standards and of the type of buffer. Kits contemplated by the invention include, but are not limited to kits for determining the amount of target nucleic acids in a biological sample, and kits determining the amount of one or more transcripts that is expected to be increased or decreased after administration of a medicament or a drug, or as a result of a disease condition such as cancer.

The present invention also provides kits specific for the detection of particular gene expression signatures, as described above. For example, a kit containing target specific bead sets for detecting microRNA for use in determining microRNA expression profiles in samples, including for example diagnostic screening kits.

EXAMPLES Example 1 A Bead-Based Gene Expression Signature Analysis Method Materials and Methods Cell Culture and RNA Isolation:

HL60 (human promyelocytic leukemia) cells were cultured in RPMI™ supplemented with 10% fetal bovine serum and antibiotics. Cells were treated with 1 μM tretinoin (all-trans-retinoic acid; SIGMA-ALDRICH™) in dimethylsulfoxide (DMSO; final concentration 0.1%) or DMSO alone for five days. Total RNA was isolated from bulk cultures with TRIzol Reagent (INVITROGEN™) in accordance with the manufacturer's directions. Cells cultured in microtiter plates were treated with 200 nM tretinoin or DMSO for two days and prepared for mRNA capture by the addition of Lysis Buffer (RNAture).

Microarrays:

Total RNA was amplified and labeled using a modified Eberwine method, the resulting cRNA hybridized to Affymetrix GeneChip HG-U133A oligonucleotide microarrays, and the arrays scanned in accordance with the manufacturer's directions. Intensity values were scaled such that the overall fluorescence intensity of each microarray was equivalent. Expression values below an arbitrary baseline (20) were set to 20. These data are provided as Tables 5-8.

Gene Selection:

The 9466 probe-sets reporting above baseline were first divided into up- and down-regulated groups by differences in mean expression levels between tretinoin and vehicle treatments. Each of these groups was further divided into three sets of approximately equal size on the basis of the lower mean expression level. The selected basal expression categories were 20-60 (low), 60-125 (moderate) and >125 (high). Probe-sets reporting small (1.5-2.5×), medium (3-4.5×) or large (>5×) changes in mean expression level within each basal expression category were extracted and ranked by signal to noise ratio. The top five probes mapping to unique RefSeq identifiers according to NetAffx in each of the eighteen categories were selected, populating nine sets of ten genes (Table 2).

Probes and Primers:

Upstream LMA probes were composed (5′ to 3′) of the complement of the T7 primer site (TAA TAC GAC TCA CTA TAG GG) (SEQ ID NO: 876), a 24 nt barcode, and a 20 nt gene-specific sequence. Downstream LMA probes were 5′-phosphorylated and contained a 20 nt gene-specific sequence and the T3 primer site (TCC CTT TAG TGA GGG TTA AT) (SEQ ID NO: 877). Barcode sequences were developed by Tm Bioscience and detailed in the FlexMAP Microspheres Product Information Sheet (LUMINEX™). Gene-specific fragments of LMA probes were designed against the Oligator Human Genome RefSet keyed by RefSeq identifier. A 40 nt region was manually selected from within these 70 nt sequences to yield two fragments of equal length with roughly similar base composition and juxtaposing nucleotides being C-G or G-C, where possible. Probe sequences are provided as Table 3. Capture probes contained the complement of the barcode sequences and had 5′-amino modification and a C12 linker. The T7 primer (5′-TAA TAC GAC TCA CTA TAG GG-3′) (SEQ ID NO: 876) was 5′-biotinylated. The T3 primer has the sequence 5′-ATT AAC CCT CAC TAA AGG GA-3′ (SEQ ID NO: 878). Oligonucleotides (all with standard desalting) were from Integrated DNA Technologies.

Beads and Bead Coupling:

xMAP Multi-Analyte COOH Microspheres (LUMINEX™) were coupled to capture probes in a semi-automated microtiter plate format. Approximately 2.5×10⁶ microspheres were dispensed to the wells of a V-bottomed microtiter plate, pelleted by centrifugation at 1800 g for 3 min, and the supernatant removed. Beads were resuspended in 25 μl of binding buffer [0.1M 2-(N-morpholino)ethansulfonic acid, pH 4.5] by sonication and pipeting, and 100 pmol of capture probe added. Two and a half μl of a freshly prepared 10 mg/ml aqueous solution of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (Pierce) was added, and the plate incubated at room temperature in the dark for 30 min. This addition and incubation step was repeated, and 180 μl 0.02% Tween-20 added with mixing. Beads were pelleted by centrifugation, as before, and washed sequentially in 180 μl 0.1% SDS and 180 μl TE (pH 8.0) with intervening spins. Coupled microspheres were resuspended in 50 μl TE (pH 8.0) and stored in the dark at 4° for up to one month. Bead mixes were freshly prepared and contained ˜1.5×10⁵/ml of each microsphere in 1.5×TMAC buffer [4.5 M tetramethylammonium chloride; 0.15% N-lauryl sarcosine; 75 mM tris-HCl, pH 8.0; 6 mM EDTA, pH 8.0]. The mapping of bead number to capture probe sequence is provided as Table 4.

Ligation-Mediated Amplification (LMA):

Transcripts were captured in oligo-dT coated 384 well plates (GenePlateHT; RNAture) from total RNA (500 ng) in Lysis Buffer (RNAture) or whole cell lysates (20 μl). Plates were covered and centrifuged at 500 g for 1 min, and incubated at room temperature for 1 h. Unbound material was removed by inverting the plate onto an absorbent towel and spinning as before. Five μl of an M-MLV reverse transcriptase reaction mix (Promega) containing 125 μM of each dNTP (INVITROGEN™) was added. The plate was covered, spun as before, and incubated at 37° for 90 min. Wells were emptied by centrifugation, as before. Ten fmol of each probe was added in 1×Taq Ligase Buffer (NEW ENGLAND BIOLABS™) (5 μl), the plate covered, spun as before, heated at 95° for 2 min and maintained at 50° for 6 h. Unannealed probes were removed by centrifugation, as before. Five μl of 1×Taq Ligase Buffer containing 2.5 U Taq DNA ligase (NEW ENGLAND BIOLABS™) was added, the plate covered, spun as before and incubated at 45° for 1 h followed by 65° for 10 min. Wells were emptied by centrifugation, as before. Fifteen μl of a HotStarTaq DNA Polymerase mix (QIAGEN™) containing 16 μM of each dNTP (INVITROGEN™) and 100 nM of T3 primer and biotinylated-T7 primer was added. The plate was covered, spun as before, and PCR performed in a THERMO ELECTRON™ MBS 384 Satellite Thermal Cycler (initial denaturation of 92° for 9 min; 92° for 30 s, 60° for 30 s, 72° for 30 s for 39 cycles; final extension at 72° for 5 min).

Hybridization and Detection:

Fifteen μl of LMA reaction product was mixed with 5 μl TE (pH 8.0) and 30 μl of bead mix (˜4500 of each microsphere) in the wells of a Thermowell P microtiter plate (Costar). The plate was covered and incubated at 95° for 2 min and maintained at 45° for 60 min. Twenty μl of a reporter mix containing 10 ng/μl streptavidin R-phycoerythrin conjugate (MOLECULAR PROBES™) in 1×TMAC buffer [3 M tetramethylammonium chloride; 0.1% N-lauryl sarcosine; 50 mM tris-HCl, pH 8.0; 4 mM EDTA, pH 8.0] was added with mixing and incubation continued at 45° for 5 min. Beads were analyzed with a LUMINEX™ 100 instrument. Sample volume was set at 50 μl and flow rate was 60 μl/min. A minimum of 100 events were recorded for each bead set and median fluorescence intensities (MFI) computed. Expression values for each transcript were corrected for background signal by subtracting the MFI of corresponding bead sets from blank (ie TE only) wells. Values below an arbitrary baseline (5) were set to 5, and all were normalized against an internal control feature (GAPDH-3′).

k-Nearest-Neighbor (KNN) Classifier:

The IVT-GeneChip data from long duration high dose tretinoin or vehicle treatments were used to train a series of KNN classifiers in the spaces of the full ninety member gene set and each of the nine ten member gene categories. These were applied to the corresponding data from the eighty-eight LMA-FlexMAP test samples whose internal reference feature (GAPDH-3′) was within two standard deviations from the mean. To permit the cross-platform analysis, both the train and test data sets were normalized so that each gene had a mean of zero and a standard deviation of one. The KNN algorithm classifies a sample by assigning it the label most frequently represented among the k nearest samples. In this case k was set to 3. The votes of the nearest neighbors were weighted by one minus the cosine distance. This analysis was performed with the GenePattern software package at world wide web address broad “dot” mit “dot” edu under cancer/software/genepattern.

Results

Measurement of seventy and eight-one transcripts has been shown to outperform established clinical and histologic parameters in disease outcome prediction for breast cancer (van de Vijver et al., 2002) and follicular lymphoma (Glas et al., 2005), respectively. Signatures of similar size and comparable prognostic power are sure to follow for a wide variety of diseases. A five member gene expression signature has also been used successfully in a cell-based small molecule screen for agents inducing the differentiation of human leukemia cells (Stegmaier et al., 2004). The absence of reliance upon prior target identification makes gene expression signature screening a powerful new strategy in drug discovery. However, immediate implementation of these and other important medical and pharmaceutical applications of genomics research is now blocked simply by the absence of a cost-effective gene expression profiling solution tailored specifically for the analysis of any feature set of up to one hundred transcripts.

High-density oligonucleotide microarrays (Lockhart et al., 1996) coupled with RNA amplification and labeling based on in vitro transcription (Van Gelder et al., 1990) provide the solution of choice for unbiased transcriptome analysis. However, the number and complexity of manipulations required, together with the cost of reagents, instrumentation, and the arrays themselves preclude its use for routine clinical and high-throughput applications. Fluorescence mediated real-time RT-PCR integrates amplification, labeling and detection Gibson et al., 1996; Morrison et al., 1998; Tyagi and Fr, 1996) and is ideal for quantitative assessment of individual transcripts. But the absence of a stable multiplex implementation makes this approach equally unsuitable for signature analysis. Conventional multiplex RT-PCR is simple and cheap but suffers from low amplification fidelity, not to mention the absence of a convenient way to detect, identify and quantify multiple amplicons.

Ligation-mediated amplification (LMA), in which two oligonucleotide probes are annealed immediately adjacent to each other on a complementary target DNA or RNA molecule and fused together by a DNA ligase (Landegren et al., 1988; Nilsson et al., 2000) to yield an synthetic amplification template (Hsuih et al., 1996), provides high targeting specificity and, by incorporating universal primer recognition sequences in fixed length ligation products, maintains target representation during multiplex PCR. Further, the ability to include distinct sequence addresses in one of the paired probes allows each of the resulting amplicons to be uniquely identified. Two gene expression profiling solutions based upon these principles—known as RASL (Yeakley et al., 2002) and RT-MLPA (Eldering et al., 2003)—each allowing the simultaneous analysis of around fifty transcripts, have been described.

The LUMINEX™ xMAP technology platform is composed of a basic auto-injecting bench-top two laser flow cytometer and a panel of one hundred sets of carboxylated polystyrene microspheres, each set being impregnated with different proportions of two fluorophores, allowing each bead to be classified on its passage through the flow cell world wide web address luminexcorp “dot” com. Furnishing bead sets with so-called molecular barcodes (Shoemaker et al., 1996)—short unique DNA sequences with uniform hybridization characteristics—delivers an optimized universal detection solution for amplicons designed to contain complementary sequences (Iannone et al., 2000). The simplicity, flexibility, throughput and modest capital and operating costs of the LUMINEX™ system compares very favorably with the self-assembled bead fiber-optic bundle array and capillary electophoresis detection pieces intrinsic to the RASL and RT-RLPA procedures (Eldering et al., 2003; Yeakley et al., 2002). This motivated evaluation of an integrated LMA-FlexMAP gene expression signature analysis solution (FIG. 1). A detailed description of our method is also available online at world wide web address broad “dot” mit “dot” edu/cancer.

A ninety member gene expression signature was derived from an unbiased genome-wide transcriptional analysis of a cell culture model of differentiation. Total RNA was isolated from HL60 cells following treatment with tretinoin or vehicle (DMSO) alone, amplified and labeled by in vitro transcription (IVT), and target hybridized to Affymetrix GeneChip microarrays. Features reporting above threshold were binned into three groups of equal size on the basis of expression level. Ten transcripts exhibiting low, moderate and high differential expression between the two conditions were then selected from each bin, populating a matrix of nine classes (Table 2) representing the diversity of expression characteristics.

Probe pairs incorporating unique FlexMAP barcode sequences were designed against each of the ninety transcripts (Table 3) and ten aliquots of the two original RNA samples were analyzed in this space by LMA-FlexMAP. Following subtraction of background signals, thresholding and normalization against an internal reference control feature (ie GAPDH), 98.5% of data points fell within two fold of their corresponding means (FIG. 2). This compares well with a similar assessment of variability for RASL (Yeakley et al., 2002) and demonstrates the high reproducibility of the method. Most of the variability was accounted for by a single feature (13/38 failures) and two wells (17/38).

There was a poor overall correlation between the mean expression levels reported by the two platforms (correlation coefficient=0.714). LMA-FlexMAP appears to overestimate transcript levels relative to IVT-GeneChip but to a degree inversely related to absolute level (FIG. 3). Estimates of the extent of differential expression reported by our solution were correspondingly less across the entire feature space, but there was broad qualitative agreement in this parameter even in the low basal and low differential expression classes (FIG. 4). Five probe pairs produced gross errors, in line with our typical first-pass probe failure rate of 5%. One failure is attributable to ambiguous annotation of the microarray and another to high background signal. All failure modes can generally be remedied by probe redesign. Irrespective, the overall correlation of log ratios between the platforms was 0.924, somewhat higher than that reported for a similar comparison between oligonucleotide and cDNA microarrays (Yuen et al., 2002). We repeated this entire LMA-FlexMAP analysis on two separate occasions with similar results. The coefficient of variation of mean expression level for each of the ninety features across all three independent evaluations had a mean of 13.8% (maximum of 49.8%), indicating high stability of the platform.

Next, we applied our method to an idealized gene expression signature analysis problem, requiring the ability to diagnose the presence of a predefined biological state in each of a large number of samples. Data were collected for our ninety gene feature set from ninety-four microtiter well cultures of HL60 cells each treated with either tretinoin or vehicle alone. Drug concentration and treatment duration were reduced by 80% and 60%, respectively, to model the sub-maximal signatures encountered in a small molecule screen. Process time from the additional of cell lysis buffer to data delivery was sixteen hours, and overall unit cost was approximately $2. Six wells (6.4%) had internal control features signals more than two standard deviations from the mean and were discarded. This throughput and overall drop out rate is typical.

Although the feature set was designed to represent the diversity of expression characteristics rather than to contain the transcripts most highly correlated with the distinction, a k-nearest-neighbor (KNN) classifier (Cover and Hart, 1967) trained on the original high dose long duration IVT-GeneChip data delivered 100% classification accuracy for these low dose short duration samples in the full ninety gene feature space. Classifiers built in the space of each of the nine ten member gene categories had error rates between 14.8% (medium level, low differential expression) and 0% (high level, high differential expression) (Table 1). These results demonstrate both the successful deployment of our solution and the advantage of a method with higher level multiplexing capability.

Our solution underestimates changes in expression level relative to the industry-standard high-end state-of-the-art gene expression profiling platform. However, its impressive classification accuracy in an idealized application indicates that performance can easily be sacrificed for throughput in pursuit of a practical gene expression signature analysis solution, and bodes well for the rapid deployment of any legacy signature with minimal or even no optimization. The assessments reported here also suggest that new signatures designed specifically for this platform should exploit the full content capacity and avoid transcripts expressed at low or moderate levels with low degrees of differential expression. With its simplicity, flexibility, throughput and cost-effectiveness the LMA-FlexMAP method has been a transformative tool in our laboratories whose exploitation for biological discovery shall be reported elsewhere.

Example 2 A Bead-Based microRNA Expression Profiling Method Materials and Methods Samples

Details of sample information are available in Table 9. Total RNAs were prepared from tissues or cell lines using TRIzol (INVITROGEN™, Carlsbad, Calif.), as described (Ramaswamy et al., 2001), and in compliance with IRB protocols. Leukemia bone marrow mononuclear cells were collected from patients treated at ST. JUDE CHILDREN'S RESEARCH HOSPITAL™ and at DANA-FARBER CANCER INSTITUTE™ and their immunophenotype and genotype determined as previously described (Ferrando et al., 2002; Yeoh et al., 2002). Normal mouse lung and mouse lung cancer samples were collected from KRasLA1 mice, and genotyped as described (Johnson et al., 2001). Lungs from four- to five-month old mice were inflated with phosphate-buffered saline prior to removal. Individual lung tumors and normal lungs were dissected and immediately frozen on dry ice before RNA preparation. HL-60 cells were plated at 1.5×10⁵ cell/ml and induced to differentiate by 1 μM all-trans retinoic acid (SIGMA™, St. Louis, Mo.; in ethanol). Cells were harvested after 1, 3 and 5 days. Culturing conditions for other cells are detailed in Example 3.

miRNA Labelling

Target preparation from total RNA follows the described procedure (Miska et al., 2004), with modifications. Briefly, two synthetic pre-labeling-control RNA oligonucleotides (5′-pCAGUCAGUCAGUCAGUCAGUCAG-3′ (Seq ID No: 872), and 5′-pGACCUCCAUGUAAACGUACAA-3′ (Seq ID No: 873), DHARMACON™, Lafayette, Colo.) were used to control for target preparation efficiency. They were each spiked at 3 fmoles per μg total RNA. Small RNAs (18- to 26-nucleotide) were recovered from 1 to 10 μg total RNA through denaturing polyacrylamide gel purification. Small RNAs were adaptor-ligated sequentially on the 3′-end and 5′-end using T4 RNA ligase (AMERSHAM BIOSCIENCES™, Piscataway, N.J.). After reverse-transcription using adaptor-specific primer, products were PCR amplified (95° C. 40 sec, 50° C. 30 sec, 72° C. 30 sec, 18 cycles for 10 μg starting total RNA; 3′-primer: 5′-tactggaattcgcggtta-3′ (Seq ID No: 874), 5′ primer: 5′-biotin-caacggaattcctcactaaa-3′ (Seq ID No: 875), IDT, Coralville, Iowa). For side-by-side comparison of the bead-detection and the glass-microarray, a 5′-Alexa-532-modified primer was used for compatibility with the glass-microarray. PCR products were precipitated and dissolved in 66 μl TE buffer (10 mM TrisHCl, pH8.0, 1 mM EDTA) containing two biotinylated post-labeling-control oligonucleotides (100 fmoles of FVR506, and 25 fmoles PTG20210, see Table 10).

Bead-Based Detection

miRNA capture probes were 5′-amino-modified oligonucleotides with a 6-carbon linker (IDT). Capture probes for miRNAs and controls were divided into three sets (see Table 10), and each sample was profiled in 3 assays on these three probe sets separately. Probes were conjugated to carboxylated xMAP beads (LUMINEX™ Corporation, Austin, Tex.) in 96-well plates, following the manufacturer's protocol. For each probe set, 3 μl of every probe-bead conjugate were mixed into 1 ml of 1.5×TMAC (4.5 M tetramethylammonium chloride, 0.15% sarkosyl, 75 mM Tris-HCl, pH 8.0, 6 mM EDTA). Samples were hybridized in a 96-well plate, with two mock PCR samples (using water as template) in each plate for background control. Hybridization was carried out with 33 μl of the bead mixture and 15 μl of labelled material, at 50° C. overnight. Beads were spun down, resuspended in 1×TMAC containing 10 μg/ml streptavidin-phycoerythrin (MOLECULAR PROBES™, Eugene, Oreg.) and incubated at 50° C. for 10 minutes before data acquisition on a LUMINEX™ 100IS machine. Median fluorescence intensity values were measured.

Computational Analyses

Profiling data were first scaled according to the post-labeling-controls and then the pre-labeling-controls, in order to normalize readings from different probe/bead sets for the same sample, and to normalize for the labeling efficiency, as detailed in Materials and Methods of Example 3. Data were thresholded at 32 and log₂-transformed. Hierarchical clustering was performed with average linkage and Pearson correlation. Prior to clustering, data were filtered to eliminate genes with expression lower than 7.25 (on log₂ scale) in all samples. Next, all features were centered and normalized to a mean of 0 and a standard deviation of 1. k-Nearest-Neighbor classification of normal vs. tumor was performed with k=3 in the selected feature space using Euclidean distance measure. Note that different metrics were used for clustering and normal/tumor classification. Features were selected for the distinction between all normal samples vs. all tumors (for colon, kidney, prostate, uterus, lung and breast; P<0.05 after Bonferroni-correction). P values were calculated using a variance-fixed t-test with a minimal standard deviation of 0.75, after confounding the tissue types. Multi-class predictions of poorly differentiated tumors were performed using the probabilistic neural network algorithm, a Gaussian-weighted nearest neighbor method. For each test sample, the tissue type that had the highest probability in multiple one-tissue-versus-the-rest predictions was assigned. Feature number and the Gaussian width were optimized based on leave-one-out cross-validations on the training data set. Features were selected based on the variance-fixed t-test score, requiring equal number of up- and down-regulated features. Distances were based on the cosine in the selected feature space.

Expression Data

miRNA expression data have been submitted to GEO at world wide web address at ncbi “dot” nih “dot” gov/geo, with a series accession number of GSE2564. mRNA expression data were published previously (Ramaswamy et al., 2001), and are available together with miRNA expression data at world wide web address broad “dot” mit “dot” edu under cancer/pub.

Results and Discussion

Much progress has been made over the past decade in developing a molecular taxonomy of cancer (see review Chung et al., 2002). In particular, it has become clear that among the ˜22,000 protein-coding transcripts are mRNAs capable of classifying a wide variety of human cancers (Ramaswamy et al., 2001). Recently, hundreds of small, non-coding miRNAs have been discovered (see review Bartel, 2004). The first identified miRNAs, the products of the C. elegans genes lin-4 and let-7, play important roles in controlling developmental timing and probably act by regulating mRNA translation (Ambros and Horvitz, 1984; Lee et al., 1993; Reinhart et al., 200). When lin-4 or let-7 is inactivated, specific epithelial cells undergo additional cell divisions as opposed to their normal differentiation. Since abnormal proliferation is a hallmark of human cancers, it seemed possible that miRNA expression patterns might denote the malignant state. Furthermore, altered expression of a few miRNAs has been found in some tumor types (Calin et al., 2002; E is et al., 2005; Johnson et al., 2005; Michael et al., 2003). However, the potential for miRNA expression to inform cancer diagnosis has not been systematically explored.

To determine the expression pattern of all known miRNAs, we first needed to develop an accurate and inexpensive profiling method. This goal is challenging, because of the miRNAs' short size (around 21 nucleotides) and the sequence similarity of members of miRNA families. Glass-slide microarrays have been used for miRNA profiling (Babak et al., 2004; Barad et al., 2004; Liu et al., 2004; Miska et al., 2004; Nelson et al., 2004; Thomson et al., 2004; Sun et al., 2004), but cross-hybridization of related miRNAs has been problematic. We therefore developed a bead-based profiling method. Oligonucleotide-capture probes complementary to miRNAs of interest were coupled to carboxylated 5-micron polystyrene beads impregnated with variable mixtures of two fluorescent dyes that yield up to 100 colors, each representing a miRNA. Following adaptor ligations utilizing both the 5′-phosphate and the 3′-hydroxyl groups of miRNAs (Miska et al., 2004), reverse-transcribed miRNAs were PCR-amplified using a common biotinylated primer, hybridized to the capture beads, and stained with streptavidin-phycoerythrin. The beads were then analyzed on a flow cytometer capable of measuring bead color (denoting miRNA identity) and phycoerythrin intensity (denoting miRNA abundance) (FIG. 5B).

Bead-based hybridization has the theoretical advantage that it may more closely approximate hybridization in solution and as such the specificity might be expected to be superior to glass microarray hybridization. Indeed, a spiking experiment involving 11 related sequences comparing bead-based detection to microarray-based detection demonstrated increased specificity of beads compared to microarrays, even for single base-pair mismatches (FIG. 6 a, 6 b). In addition, the bead method exhibited linear detection over two logs of expression (Example 3). Eight miRNAs were validated by northern blotting in seven cell lines. In all cases, bead-based detection paralleled the northern data (FIG. 6 c). These results demonstrate that bead-based miRNA detection is feasible, having the attractive properties of improved accuracy, high speed and low cost. The bead-based detection platform also provides flexibility in that additional miRNA capture beads can be added to the mixture, thereby detecting newly discovered miRNAs.

We then set out to determine the expression pattern of all known miRNAs across a large panel of samples representing a diversity of human tissues and tumor types. While miRNA expression has been previously explored in small sets of tissues (Babak et al., 2004; Barad et al., 2004; Liu et al., 2004; Nelson et al., 2004; Thomson et al., 2004; Sun et al., 2004) or isolated cell types (e.g. chronic lymphocytic leukemia in Calin et al., 2001), the extent of differential expression of miRNAs across cancers has not been previously determined. Indeed, one might not have expected that miRNA expression patterns would be informative with respect to cancer diagnosis, because of the relatively small number of miRNAs encoded in the genome. Remarkably, we observed differential expression of nearly all miRNAs across cancer types (FIG. 7 a). Moreover, hierarchical clustering of the samples in the space of miRNAs recapitulated the developmental origin of the tissues. For example, samples of epithelial origin fell on a single branch of the dendrogram, whereas the other major branch was predominantly populated with hematopoietic malignancies.

Furthermore, the miRNAs partitioned tumors within a single lineage. For example, we examined the miRNA profiles of 73 bone marrow samples obtained from patients with acute lymphoblastic leukemia (ALL). As shown in FIG. 7 b, hierarchical clustering revealed non-random partitioning of the samples into three major branches: one containing all 5 t(9;22) BCR/ABL positive ALLs and 10 of 11 t(12;21) TEL/AML1 cases, a second branch containing 15/19 T-cell ALLs, and a third containing all but one of the samples with MLL gene rearrangement. These experiments demonstrate that even within a single developmental lineage, distinct patterns of miRNA expression reflecting mechanism of transformation are observable and further support the notion that miRNA expression patterns encode the developmental history of human cancers.

Among the epithelial samples, those of the gastrointestinal tract were of particular interest. Samples from colon, liver, pancreas and stomach all clustered together (FIG. 7 a), reflecting their common derivation from tissues of embryonic endoderm. That is, the dominant structure in the space of miRNAs was one of developmental history. In contrast, when these samples were profiled in the space of ˜16,000 mRNAs, the coherence of gut-derived samples was not recovered (FIG. 7 c). This observation may result from the large amount of noise and unrelated signals that are embedded in the high dimensional mRNA data. Whether or not the miRNAs that are highly expressed in the gut-associated cluster (miR-192, miR-194, miR-215) play a functional role in the specification of gut development or gut-derived tumors remains to be investigated.

Having determined that miRNA expression distinguishes tumors of different developmental origin, we next asked whether miRNAs could be used to distinguish tumors from normal tissues. We previously reported that there exist no robust mRNA markers that are uniformly differentially expressed across tumors and normal tissues of different lineages (Ramaswamy et al., 2001). It was therefore striking to observe that despite the fact that some miRNAs are upregulated or unchanged, the majority of the miRNAs (129/217, p<0.05, after correction for multiple hypothesis testing) had lower expression in tumors compared to normal tissues, irrespective of cell type (FIG. 8 a). Importantly, the cancer cell lines also showed low miRNA expression relative to normal tissues (FIG. 9).

To exclude any possibility that the differential miRNA expression might be related to differences in collection of tumor vs. normal samples, we studied a mouse model of KRas-induced lung cancer (Johnson et al., 2001). We isolated miRNAs from normal lung or lung adenocarcinomas from individual mice, thereby precluding any differences in collection procedure. Notably, because of miRNA sequence conservation between human and mouse, the same miRNA capture beads could be used to profile the murine samples. As shown in FIG. 8 b, the same tumor vs. normal distinction is seen in the mouse. Accordingly, a tumor-normal classifier built on human samples had 100% accuracy when tested in the mouse. Taken together, these studies indicate that miRNAs are unexpectedly rich in information content with respect to cancer.

Our observation that miRNA expression appeared globally higher in normal tissues compared to tumors led to the hypothesis that global miRNA expression reflects the state of cellular differentiation. To test this hypothesis, we explored an experimental model in which we treated the myeloid leukemia cell line HL-60 with all-trans retinoic acid, a potent inducer of neutrophilic differentiation (Stegmaier et al., 2004). As predicted, miRNA profiling demonstrated the induction of many miRNAs coincident with differentiation (FIG. 8 c). In primary human hematopoietic progenitor cells undergoing erythroid differentiation in vitro, we observed a similar increase in miRNA expression occurring at a stage in differentiation when the cells continued to proliferate (see Example 3). These experiments support the hypothesis that global changes in miRNA expression are associated with differentiation, the abrogation of which is a hallmark of all human cancers. These findings are also consistent with the recent observation that mouse embryonic stem cells lacking Dicer, an enzyme required for miRNA maturation, fail to differentiate normally (Kanellopoulou et al., 2005).

We next turned to a more challenging diagnostic distinction: that of tumors of histologically uncertain cellular origin. It is estimated that 2%-4% of all cancer diagnoses represent cancers of unknown origin or diagnostic uncertainty (see review Pavlidis et al., 2003). To address this, we analyzed 17 poorly differentiated tumors whose histological appearance alone was non-diagnostic, but whose clinical diagnosis was established by anatomical context, either directly (e.g. a primary tumor arising in the colon) or indirectly (a metastasis of a previously identified primary). A training set of 68 more-differentiated tumors representing 11 tumor types for which both mRNA and miRNA profiles were available was used to generate a classifier. This classifier was then used without modification to classify the 17 poorly-differentiated test samples. As a group, poorly differentiated tumors had lower global levels of miRNA expression compared to the more-differentiated training set samples (FIG. 10), consistent with the notion that miRNA expression is closely linked to differentiation. Despite this overall low level of miRNA expression, the miRNA-based classifier established the correct diagnosis of the poorly differentiated samples far beyond what would be expected by chance for an 11-class classifier (12/17 correct; p<5×10⁻¹¹). In contrast, the mRNA-based classifier was highly inaccurate (1/17 correct; p=0.47), as we previously reported (Ramaswamy et al., 2001).

The experiments reported here demonstrate the feasibility and utility of monitoring the expression of miRNAs in human cancer. The unexpected findings are the extraordinary level of diversity of miRNA expression across cancers and the large amount of diagnostic information encoded in a relatively small number of miRNAs. The implication is that, unlike with mRNA expression, a modest number of miRNAs (˜200 in total) might be sufficient to classify human cancers. Moreover, the bead-based miRNA detection method has the attractive property of being not only accurate and specific but also being easily implementable in a routine clinical setting. In addition, unlike mRNAs, miRNAs remain largely intact in routinely collected, formalin-fixed paraffin-embedded clinical tissues (Nelson et al., 2004). More work is required to establish the clinical utility of miRNA expression in cancer diagnosis, but the work described here indicates that miRNA profiling has unexpected diagnostic potential. The mechanism by which miRNAs are under-expressed in cancer remains unknown. We did not observe substantive decreases of mRNAs encoding components of the miRNA processing machinery (Dicer, Drosha, Argonaute2, DGCR8 (Cullen, 2004), Example 3), but clearly other mechanisms of regulating miRNAs are possible.

The findings reported here are consistent with the hypothesis that in mammals, as in C. elegans, miRNAs can function to prevent cell division and drive terminal differentiation. An implication of this hypothesis is that down-regulation of some miRNAs might play a causal role in the generation or maintenance of tumors. Epithelial cells affected in C. elegans lin-4 and let-7 miRNA mutants generate a stem-cell-like lineage, dividing to produce daughters that, like themselves, divide rather than differentiate (Ambros and Horvitz, 1984; Reinhart et al., 2000). We speculate that aberrant miRNA expression might similarly contribute to the generation or maintenance of “cancer stem cells” recently proposed to be responsible for cancerous growth in both leukemias and solid tumors (Al-Hajj et al., 2003; Lapidot et al., 1994; Reya et al., 2001; Singh et al., 2004).

Example 3 MicroRNA Expression Profiles Classify Human Cancers

Additional information about the paper and a frequently-asked-questions (FAQ) page are available at ______.

Materials and Methods Cell Culture

HEL, TF-1, PC-3, MCF-7, HL-60, SKMEL-5, 293 and K562 cells were obtained from the AMERICAN TYPE CULTURE COLLECTION™ (ATCC™, Manassas, Va.), and cultured according to ATCC™ instructions. All T-cell ALL cell lines were cultured in RPMI™ medium supplemented with 10% fetal bovine serum. CCRF-CEM and LOUCY cells were obtained from ATCC™. ALL-SIL, HPB-ALL, PEER, TALL1, P12-ICHIKAWA cells were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). SUPT11 cells were a kind gift of Dr. Michael Cleary at Stanford University.

Umbilical cord blood was obtained under an IRB approved protocol from the Brigham and Women's Hospital. Light-density mononuclear cells were separated by Ficoll-Hypaque centrifugation, and CD34⁺ cells (85-90% purity) were enriched using Midi-MACS columns (Miltenyi Biotec, Auburn, Calif.). Erythroid differentiation of the CD34⁺ cells was induced in two stages in liquid culture (Ebert et al., 2005). For the first seven days, cells were cultured in Serum Free Expansion Medium (SFEM, Stem Cell Technologies, Tukwila, Wash.) supplemented with penicillin/streptomycin, glutamine, 100 ng/mL stem cell factor (SCF), 10 ng/mL interleukin-3 (IL-3), 1 μM dexamethasone (SIGMA™), 40 μg/ml lipids (SIGMA™), and 3 IU/ml erythropoietin (Epo). After 7 days, cells were cultured in the same medium without dexamethasone and supplemented with 10 IU/ml Epo. For flow cytometry analyses, approximately 1 to 5×10⁵ cells were labeled with a phycoerythrin-conjugated antibody against glycophorin-A (CD235a, Clone GA-R2, BD-PHARMINGEN™, San Jose, Calif.) and a FITC-conjugated antibody against CD71 (Clone M-A712, BD-PHARMINGEN™). Flow cytometry analyses were performed using a FACScan flow cytometer (BECTON DICKINSON™).

Glass-Slide Detection of miRNAs

Glass slide microarrays were spotted oligonucleotide arrays and hybridized as described previously (Miska et al., 2004). Briefly, 5′-amino-modified oligonucleotide probes (the same ones as used on the bead platform) were printed onto amide-binding slides (CodeLink, AMERSHAM BIOSCIENCES™). Printing and hybridization were done following the slides manufacturer's protocols with the following modifications: oligonucleotide concentration for printing was 20 μM in 150 mM sodium phosphate, pH 8.5. Printing was done on a MicroGrid TAS II arrayer (BioRobotics) at 50% humidity. Labeled PCR product was resuspended in hybridization buffer (5×SSC, 0.1% SDS, 0.1 mg/ml salmon sperm DNA) and hybridized at 50° C. for 10 hours. Microarray slides were scanned using an arrayWoRx^(e) biochip reader (APPLIED PRECISION™) and primary data were analyzed using the Digital Genome System suite (MOLECULARWARE™).

Northern Blot Analysis

Northern blot analyses were carried out as described (Lau et al., 2001). Total RNAs from cell lines were loaded at 10 μg per lane. Blots were detected with DNA probes complementary for human miR-20, miR-181a, miR-15a, miR-16, miR-17-5p, miR-221, let-7a, and miR-21.

Quantitative RT-PCR

Reverse transcription (RT) reactions were carried out on 50 to 200 ng total RNA in 10 μl reaction volumes, using the TAQMAN™ reverse transcription kit (APPLIED BIOSYSTEMS™, Foster City, Calif.) and random hexamers, following the manufacturer's protocol. RT products were diluted 5-fold in water and assayed using TAQMAN™ Gene Expression Assays (APPLIED BIOSYSTEMS™) in triplicates, on an ABI PRISM 7900HT real-time PCR machine. Efficiency of PCR amplification was determined by 5 two-fold-serial-diluted samples from HL-60 cDNA. The TAQMAN™ Gene Expression Assays used are listed in the parentheses. (Dicer1: Hs00998566_m1; Ago2/EIF2C2: Hs00293044_m1; Drosha/RNase3L: Hs00203008_m1; DGCR8: Hs00256062_m1; and eukaryotic 18S rRNA endogenous control)

Data Preprocessing and Quality Control

To eliminate bead-specific background, the reading of every bead for every sample was first processed by subtracting the average readings of that particular bead in the two-embedded mock-PCR samples in each plate. As stated in the Methods, every sample was assayed in three wells. Each of the three wells contained 94 probes (19 common probes and 75 unique ones). Out of the 19 common probes are the two pre-labeling controls and the two post-labeling controls. Quality control was performed as part of the preprocessing by requiring that the reading from each control probe exceeds some minimal probe-specific threshold. These thresholds were determined by identifying a natural lower cutoff, i.e. a dip, in the distribution of each control probe. The cutoff values were chosen based on a set of samples in a pilot study. The lower post-control should be greater than 500 and the higher post-control must exceed 2450. The lower and higher pre-controls should exceed 1400 and 2000 respectively (after well-to-well scaling). In this study, about 70% of the samples passed the quality control. Note that the above specifications were used on version 1 of the platform. A similar preprocessing was performed on version 2 of the platform.

Preprocessing was done in four steps: (i) well-to-well scaling—the reading from each well were scaled such that the total of the two post-labeling controls, in that well, became 4500 (a median value based on a pilot study); (ii) sample scaling—the normalized readings were scaled such that total of the 6 pre-labeling controls in each sample reached 27,000 (a median value based on a pilot study); (iii) thresholding at 32 (see below); and (iv) log₂ transformation. All control probes, as well as a probe (EAM296) which had a high background in the absence of any prepared target, were removed before any further analysis. After eliminating these probes, 217 (255 for version 2 of the platform) features were left and these were used throughout the analysis.

Hierarchical Clustering

miRNA expression data first underwent filtering. The purpose of this filtering is to remove features which have no detectable expression and thus are uninformative but may introduce noise to the clustering. A miRNA was regarded as “not expressed” or “not detectible”, if in none of the samples, that particular miRNA has an expression value above a minimal cutoff. We applied a cutoff of 7.25 (after data were log₂-transformed). This cutoff value was determined based on noise analyses of target preparation and bead detection (see below and FIG. 12 a). In that experiment, the majority of features had a standard deviation below 0.75 when their mean was over 5 in log₂-transformed data. Thus we used a cutoff of 3 standard deviations above the minimal expression level (5+3×0.75=7.25). Any feature that is not expressed under this criterion was filtered out before clustering. Data were then centered and normalized for each feature, bringing the mean to 0 and the standard deviation to 1. This equalizes the contributions of all features. For hierarchical clustering, we used Pearson correlation as a similarity measure, and used the average-linkage algorithm (Jain et al., 1988) for both the samples and the features.

k-Nearest Neighbor (kNN) Prediction

After feature filtration (described in the hierarchical clustering), marker selection was performed on 187 features. The variance-thresholded t-test score was used as a measure to score features. A minimal standard deviation of 0.75 was applied. Markers were searched among the filtered miRNAs. Nominal P-value was calculated for each feature, by permuting the class labels of the samples. In order to select features that best distinguish tumors from normal samples on all tissue types, i.e. taking into account the confounding tissue-type phenotype, restricted permutations were performed (Good, 2004). In restricted permutations, one shuffles the tumor/normal labels only within each tissue type to get the distribution under the desired null hypothesis. To achieve accurate estimates for the p-values, 400 times the number of features (400×187=74,800) of iterations were performed. To correct for multiple-hypotheses testing, markers were selected requiring the Bonferroni-corrected P-values to be less than 0.05. kNN prediction was performed using the kNN module in the GenePattern software, with k=3 and a Euclidean distance measure (GenePattern at ______).

Probabilistic Neural Network (PNN) Prediction

A two-class PNN (Specht, 1990) prediction was calculated based on the following class posterior probability:

${{P\left( c \middle| x \right)} = {\frac{{P\left( x \middle| c \right)}{P(c)}}{\sum\limits_{c^{\prime}}{{P\left( x \middle| c^{\prime} \right)}{P\left( c^{\prime} \right)}}} = \frac{\frac{P(c)}{n_{c}}{\sum\limits_{i:{{\overset{\_}{y}}_{i} \in c}}{\exp \left( {{{- {D\left( {x,y_{i}} \right)}^{2}}/2}\sigma^{2}} \right)}}}{\sum\limits_{c^{\prime}}\left\lbrack {\frac{P\left( c^{\prime} \right)}{n_{c^{\prime}}}{\sum\limits_{i:{{\overset{\_}{y}}_{i} \in c^{\prime}}}{\exp \left( {{{- {D\left( {x,y_{i}} \right)}^{2}}/2}\sigma^{2}} \right)}}} \right\rbrack}}},$

where x is the predicted sample and c is the class for which the posterior probability is calculated. The training set samples are y_(i), n_(c) is the number of samples of class c in the training set, and D(x,y_(i)) is the distance between the predicted sample and training sample i. In our case, the sum in the denominator (of c′) is over two class values, since we predict a sample either to belong or not to belong to a specific tissue-type. Note that the first step is derived using Bayes rule which allows to incorporate a prior probability for each class, P(c). We used a uniform prior over all 11 tissue-types which translated to 1/11 for being in a certain type and 10/11 for not being in that type. We did not use the tissue-type frequencies in the training set since they likely do not represent the frequencies of different tumors in the general population.

Multi-class prediction using PNN was achieved by breaking down the question into multiple one vs. the rest (OVR) predictions. To perform PNN OVR two-class classification, we built a model based on the training set. This model has two parameters: the number of features used, and σ (the standard deviation of the Gaussian kernel which is used to calculate the contribution of each training sample to the classification). The optimal parameters (for each OVR classifier) were selected using a leave-one-out cross-validation procedure from all possible parameter-pairs in which the number of features ranges from 2 to 30 in steps of 2 and σ takes the values from 1 to 4 times the median nearest neighbor distance, in steps of 0.5 (a total number of 105 combinations). The best model was determined by (i) the fewest number of leave-one-out errors on the training set, which include both false-positive and false-negative errors with the same weight, and (ii) among all conditions with the same error rate, the parameters that gave rise to the maximal mean log-likelihood of the training set were selected. The mean log-likelihood is defined

as

${L\left\lbrack {\left\{ x_{i} \right\};M} \right\rbrack} = {\frac{1}{\# \mspace{14mu} {of}\mspace{14mu} {training}\mspace{14mu} {examples}}{\sum\limits_{i}{\log \left( {P_{M}\left( c_{i} \middle| x_{i} \right)} \right)}}}$

where c_(i) is the true class of sample x, and the probability is evaluated using the model M. The top n features were selected using the variance-thresholded t-test score in a balanced manner; n/2 features with the top positive scores and n/2 features with most negative scores. The cosine distance measure was used; D(x,y_(i))=1-cosine(x,y_(i)).

P-Value Calculation for the Number of Correct Classifications

A Binomial distribution was used to calculate the probability to obtain at least the number of correct classifications (on the test set) as we observed. Assuming a random classifier would predict the tissue-type randomly with a uniform distribution over the 11 possible outcomes, the probability of a correct classification is 1/11. This is applicable to the PNN prediction, in which the background frequency of each tissue type was assumed to be 1/11. The p-value is, therefore, the tail of the Binomial distribution from the observed number of correct classifications, s, to the total number of samples in the test set, n:

${P - {value}} = {\sum\limits_{t = s}^{n}{\begin{pmatrix} n \\ t \end{pmatrix}{p^{t}\left( {1 - p} \right)}^{n - t}}}$

where p is one over the number of tissue-types (1/11, in our case) and t is the number of correct classification which goes from the observed number, s, to the maximum of possible correct samples n.

Results and Discussion

Development of a Bead-Based miRNA Profiling Platform

Compared with glass-based microarrays, bead-based profiling solutions have the advantages of higher sample throughput and liquid phase hybridization kinetics, while having the disadvantage of lower feature throughput. For the genomic analysis of miRNA expression, this disadvantage is negligible because of the relative small number of identified miRNAs. Since new miRNAs are still being discovered, the flexibility and ease of these “liquid chips” to introduce new features is of particular value.

We developed a bead-based miRNA profiling platform, as detailed in the Methods section. Version 1 of this platform (used for most samples in this study) covers 164 human, 185 mouse, and 174 rat miRNAs, according to Rfam 5.0 miRNA registry database (Ambros et al., 2003; Griffiths-Jones, 2004). Version 2 of this platform (used for the acute lymphoblastic leukemia study and the erythroid differentiation study) covers additional 24 human, 13 mouse and 2 rat miRNAs (refer to Table 10 for details).

This profiling platform is compatible in theory with any miRNA labeling method that labels the sense strand. For our study, we followed one described by Miska et al., 2004 that labels mature miRNAs through adaptor ligation, reverse-transcription and PCR amplification. We reasoned that the amplification step will allow future use of these labeled materials, which were from precious clinical samples. Defined amounts of synthetic artificial miRNAs were added into each sample of total RNAs as pre-labeling controls. This allows us to normalize the profiling data according to the starting amount of total RNA, using readings from capture probes for these synthetic miRNAs (see Methods for details). This contrasts the use of total feature intensity to normalize the readings of different samples; the hidden assumption of the latter is that the total miRNA expression is the same in all samples, which may not be true considering the small known number of miRNAs.

We analyzed the variation caused by labeling and detection using repetitive assays of the same RNA samples of a few cell lines originated from different tissues; these cell lines have different miRNA profiles. We plotted the standard deviation of each probe versus its means, after the data were log₂-transformed (FIG. 12 a). The variations are large for low means, and decrease and stabilize with increasing means. For most measured features with mean above 5 (32 before log₂-transformation), the standard deviation is below 0.75. This value of mean provides a good cutoff for a lower threshold of the data, which was thus used in this study.

We compared the data from expression profiles and northern blots on a panel of 7 cell lines; the same quantities of the same starting total RNAs were used for both analyses. We picked eight miRNAs that are expressed in any of these cell lines and that show differential expression according to the expression profiles, and probed them with northern blots. All eight display good concordance between the two assays (FIG. 6 c), indicating that our profiling platform has good accuracy.

We next examined the linearity of profiling (both labeling and detection) by measuring a series of starting materials, covering 0.5 μg to 10 μg of total RNAs from HEL cells. Most miRNAs report good linearity up to 3500 median fluorescence intensity readings (after normalization with pre-labeling-controls, FIG. 12 b). Taken together with the threshold level of 32, the profiling method has roughly 100-fold of dynamic range.

One common issue that affects hybridization-based analyses for miRNAs is the specificity of detection, since many miRNAs are closely-related on the sequence level. To assess the specificity of detection, we synthesized oligonucleotides corresponding to the reverse-transcription products of adaptor-ligated miRNAs, in this case the human let-7 family of miRNAs and a few artificial mutants. The sequences for these oligonucleotides are in Table 11, and the alignment of human let-7 miRNAs and mutant sequences are listed in Table 12. They were then labeled through PCR using the same primer sets. This provides a collection of sequence-pairs that differ by one, two, or a few nucleotides (FIG. 11 and Table 12). Results are presented in Example 2 and in FIG. 6 a,b.

Hierarchical Clustering of Multiple Cancer and Normal Samples

We applied this miRNA profiling platform for 140 human cancer specimens, 46 normal human tissues, and various cell lines. The collection of samples covers more than ten tissues and cancer types. This collection was referred to as miGCM (for miRNA Global Cancer Map). We first examined the miRNA expression profiles to see whether we can detect previously reported tissue-restricted expression of miRNAs. Indeed, we observed tissue-restricted expression patterns. For example, miR-122a, a reported liver-specific miRNA (Lagos-Quintana et al., 2002), is exclusively expressed in the liver samples, whereas miR-124a, a brain-specific miRNA (Lagos-Quintana et al., 2002), is abundantly expressed in the brain samples.

We performed hierarchical clustering on this data set, as described in the Methods. Hierarchical clustering is an unsupervised analysis tool that captures internal relationship between the samples. It organizes the samples (or features) into a tree structure (a dendrogram) according to the similarity between the samples (or the features). Close pairs of samples (ones with similar expression profiles) will generally be connected in the dendrogram at an earlier phase, while samples with larger distances (with less similar expression profiles) will be connected at a later phase (details can be found in Duda et al., 2000). The detailed result of hierarchical clustering on both the samples and features using correlation metrics is presented in FIG. 7 a and FIG. 9.

Comparison of miRNA and mRNA Clustering in Regard to GI Samples

After finding that the gastrointestinal tract samples were clustered together (Example 2 and FIG. 7 a), we asked whether or not this structure is similarly displayed by clustering in the mRNA space. We took 89 epithelial samples that have both successful mRNA and miRNA profiling data, and subjected them to hierarchical clustering. Both data underwent identical gene filtering, i.e. a lower threshold filter to eliminate genes that do not have expression values over 7.25 (on log₂ scale) in any sample, and underwent the same clustering procedure. This gene filtering resulted in 195 miRNAs and 14546 mRNAs. Data were presented in the main text, FIG. 7 c and FIG. 13. Results show that the mRNA clustering does not recover the coherence of GI samples, as identified in the miRNA expression space. Of note, the exact outcome of hierarchical clustering is dependent on the collection of samples present for analysis. Consequently, the cluster of the GI samples in miRNA clustering in FIG. 7 c is slightly different from that of FIG. 7 a, since the latter comprises of many more samples.

In order to test whether the lack of coherence of GI samples in the mRNA clustering is sensitive to the choice of genes that were used to represent each sample, we tested two additional gene filtering methods. First, we used a variation filter as was performed in Ramaswamy et al., 2001 (lower threshold of 20, upper threshold of 16000, the maximum value is at least 5 fold greater than the minimum value, and the maximum value is more than 500 greater than the minimum value), which yielded 6621 genes. Second, we examined only transcription factors, a set of gene regulators as are miRNAs. We took the genes that passed the above variation filter and that are also annotated with transcription factor activity in the Gene Ontology (GO:0003700). This resulted in 220 transcription factors as listed in the Table 13. Similar to the minimum-expression filter on the mRNA data, these two gene selection methods yielded clustering by tissue types to a certain degree. However, none recovered the gut coherence (FIG. 13). This indicated either that the miRNA space contains some different information from the mRNA space or that in the mRNA space, the gut signal is masked by other signals or noise. Importantly, a set of transcription factors did not mimic miRNAs in this test, suggesting the difference is not solely due to the gene regulator nature of miRNAs.

Normal/Tumor Classifier and kNN Prediction of Mouse Lung Samples

In order to build a classifier of normal samples vs. tumor samples based on the miGCM collection, we first picked tissues that have enough normal and tumor samples (at least 3 in each class). Table 14 summarizes the tissues for this analysis.

kNN (Duda et al., 2000) is a predicting algorithm that learns from a training data set (in this case, the above samples from the miGCM data set) and predicts samples in a test data set (in this case, the mouse lung sample set). A set of markers (features that best distinguishes two classes of samples, in this case, normal vs. tumor) was selected using the training data set. Distances between the samples were measured in the space of the selected markers. Prediction is performed, one test sample at a time, by: (i), identifying the k nearest samples (neighbors) of the test sample among the training data set; and (ii) assigning the test sample to the majority class of these k samples.

We first selected markers that best differentiate the normal and tumor samples (see Materials and Methods above) out of the 187 features that passed the filter (which was applied on the training set alone). This generated a list of 131 markers that each has a p-value <0.05 after Bonferroni correction; 129/131 markers are over-expressed in normal samples, whereas 2/131 are over-expressed in the tumor samples. Table 15 lists these markers.

These 131 markers were used without modification to predict the 12 mouse lung samples using the k-nearest neighbor algorithm. Each mouse sample was predicted separately, using log₂ transformed mouse and human expression data. The tumor/normal phenotype prediction of a mouse sample was based on the majority type of the k nearest human samples using the chosen metric in the selected feature space. Since the tumor/normal distinction was observed at the raw miRNA expression levels, we decided to use Euclidean distance to measure the distances between samples. Thus, we performed kNN with the Euclidean distance measure and k=3, resulting in 100% accuracy. The detailed prediction results are available in Table 16. Similar classification results were obtained with other kNN parameters, with the exception of one mouse tumor T_MLUNG_(—)5 (3rd column from right in FIG. 12 b). This sample was occasionally classified as normal, for example, when using cosine distance measure (k=3). It should be pointed out that cosine distance captures less an overall shift in expression levels compared to Euclidean distance. It rather focuses on comparing the relationships among the different miRNAs So it appears that the same miRNA data capture different information with different distance metrics; Pearson correlation captures information about the lineage (as seen in clustering results), and Euclidean distance captures the normal/tumor distinction.

Differentiation of HL-60 Cells

One hypothesis for the global decrease of miRNA expression in tumors (FIG. 7 a, FIG. 8 a,b) is that many miRNAs are upregulated during differentiation. We examined an in vitro differentiation system, the differentiation of HL-60 acute myeloblastic leukemia cells. HL-60 cells differentiate with increasing neutrophil characteristics upon treatment with all-trans retinoic acid (ATRA) during a course of 5 days (Stegmaier et al., 2004). We found 59 miRNAs commonly expressed (see Materials and Methods for the definition of “expressed”) in three independent experiments of HL-60 cells with or without ATRA treatment. These 59 miRNAs are shown in Table 17. A heatmap is shown in FIG. 8 c, reflecting averages of successfully profiled same condition samples. Results indicate increased expression of many miRNAs after 5 days of ATRA-induced differentiation (5d+). Since HL-60 is a cancerous cell line, this result supports the hypothesis that the global miRNA downregulation in cancer is related to differentiation. Whether or not the observed global miRNA expression change is associated with certain windows of differentiation needs further investigation.

Erythroid Differentiation of Primary Hematopoietic Cells In Vitro

We profiled the expression of miRNAs during erythroid differentiation in vitro to ask whether the increase in miRNA expression observed in the differentiation of HL-60 cells also occurs in primary cells. The accessibility of normal hematopoietic progenitor cells and the ability to recapitulate erythropoiesis in vitro provide a model to study normal differentiation. We purified CD34⁺ hematopoietic progenitor cells from umbilical cord blood. Erythroid differentiation was induced in vitro using a two phase liquid culture system. The state of differentiation of cultured cells was monitored every other day by evaluating expression of CD71 and glycophorin A (Gly-A) (FIG. 14 b). CD71 expression increases early in erythroid differentiation and gradually decreases in terminal erythroid differentiation. Gly-A expression increases later in erythropoiesis and remains elevated through terminal differentiation. As in HL60 cells, the expression of many miRNAs increased during differentiation (FIG. 14 c). Unlike HL-60 cells, the erythroid cells continued to proliferate at the time points when miRNA expression increased (FIG. 14 a). This suggests that proliferation itself, which is often integrally linked to differentiation, cannot account completely for the increased miRNA expression during differentiation.

Analyzing Tissue Samples Using an mRNA Proliferation Signature

It is conceivable that differences in cellular proliferation, often integrally linked to differentiation, may contribute to the global miRNA signals. We asked whether the miRNA global expression differences among samples are merely a consequence of their differences in proliferation rates. To estimate the proliferation rates in tissue samples, we assembled a consensus mRNA signature of proliferation, reported to positively correlate with proliferation or mitotic index in breast tumors, lymphomas and HeLa cells (Alizadeh et al., 2000; Perou et al., 2000; Whitfield, et al., 2002). Table 18 summarizes this list.

We first asked whether the mRNA proliferation signature reflects proliferation rates in our samples. Indeed, we noticed that the mean expression of these mRNAs is higher in tumors than normal tissues (FIG. 15), reflecting faster proliferation rates in tumor samples.

Next, we examined in the tumor samples the expression of the mRNA proliferation signature. We focused on lung and breast, two tissues that we have sufficient numbers of poorly differentiated tumors and more differentiated tumors. It is important to point out that poorly differentiated tumors have globally lower miRNA expression than more differentiated tumors. However, we did not observe any difference in the mRNA proliferation signature between these two categories of samples (FIG. 15). This result also suggests that the global miRNA expression is unlikely to be solely dependent on proliferation rates.

RT-PCR Analyses of Genes Involved in miRNA Machinery

One possible mechanism of the observed global miRNA expression difference between normal samples and tumors is changes in expression levels of miRNA processing enzymes. In lung cancer, Dicer levels were reported to correlate with prognosis (Karube et al., 2005). We decided to examine Dicer1, Drosha, DGCR8 and Argonaute 2 (Ago2), which are critical in miRNA processing (Tomari et al., 2005). Lacking probe sets representing these genes in our mRNA data, we used quantitative RT-PCR and analyzed 79 samples (32 normal samples and 47 tumors, covering 8 tissues, including colon, breast, uterus, lung, kidney, pancreas, prostate and bladder). We normalized the quantitative PCR data with 18S rRNA levels. We performed Student's t-test (two-tail, unequal variance) for normal/tumor phenotypes on all samples examined (P=0.3 for Dicer1, P=0.11 for Drosha, P=0.0011 for DGCR8, P=0.0138 for Ago2). DGCR8 and Ago2 have significant nominal p-values under the above test. However, the fold differences of DGCR8 and Ago2 are small between tumors and normal samples (tumor samples have higher mean threshold cycle (Ct) values for these two genes; the mean Ct differences between normal and tumor samples are: 0.776 for DGCR8 and 0.798 for Ago2, corresponding to 1.7-fold and 1.5-fold absolute level differences respectively, after correction for PCR amplification efficiency). Whether or not the observed weak decreases on the transcript level may account for the differences in miRNA expression needs further investigation. It is also important to note that these results do not exclude the possibility that these miRNA machinery genes are involved in regulating tumor/normal miRNA expression in certain cancer types, or are regulated on the protein and activity levels.

Analyses of Poorly Differentiated Tumors

We first set out to determine whether poorly differentiated tumors show a globally weaker miRNA expression than tumor samples in the miGCM collection, which represent more differentiated states. To this end, we made a comparison of poorly differentiated tumors to more differentiated tumors of the corresponding tissue types. The analysis was performed on 180 features, after the data were filtered to eliminate non-expressing miRNAs on the 55 samples which belong to tissue types that have both more-differentiated and poorly-differentiated samples (see the hierarchical clustering section in Supplementary Methods for data filtration). FIG. 10 shows that poorly differentiated tumors indeed have globally lower miRNA expression. Out of the 180 features, 95 miRNAs display lower mean expression levels in poorly differentiated tumors (p<0.05 with a variance-thresholded t-test).

We used PNN for prediction of tissue origin of poorly differentiated tumors. PNN is a probability based prediction algorithm and can be considered as a smooth version of kNN. For a multi-class prediction, PNN avoids the ambiguity often encountered with kNN, when multiple training classes are equally presented in the k nearest neighbors of a test sample. For a two-class classification problem, PNN assigns a probability for a test sample to be classified into one of the two classes. The contribution of each training sample to the classification of a test sample is related to their distance and follows the Gaussian distribution: the closer the test sample, the larger the contribution. The probability for a test sample to belong to a certain class is the total contribution from every training sample belonging to that class, divided by the total contributions of all training samples (see Materials and Methods for more details).

For the prediction of poorly differentiated tumors, the training sample set consists of 68 tumor samples with both miRNA and mRNA profiling data, covering 11 tissue types. The test set contains 17 poorly differentiated tumors. Table 19 summarizes the information on the 17 poorly differentiated tumors. To solve this multi-class prediction problem, we broke down the task into 11 two-class predictions. Each two-class prediction assigns a probability for a test sample to belong to a certain tissue-type vs. the rest of the tissue-types (one vs. the rest, OVR), for example, colon vs. non-colon. After performing OVR classifications for all 11 tissues, the one tissue-type that receives the highest probability marks the predicted tissue type. The prediction results are summarized in Table 20.

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TABLE 1 Classification Accuracy. differential expression 1.5-2.5x 3-4.5x >5x basal 20-60 12.5 2.3 2.3 expression  60-125 14.8 1.1 5.7 level >125 1.1 1.1 0 Error rates (%) of a k-nearest-neighbor classifier trained on IVT-GeneChip data to predict the true identity (tretinoin or DMSO) of eighty-eight test samples in the space of each of the nine gene classes from FIG. 4.

TABLE 2 Gene Selection mean expression log10 signal to Affymetrix level fold (fold standard deviation noise ID RefSeq ID(s) DMSO tretinoin change change) DMSO tretinoin ratio basal expression level 20-60 units fold change 1.5-2.5 200721_s_at NM_005736 51.20 81.30 1.59 0.20 1.05 1.37 12.47 210944_s_at NM_000070 52.48 130.88 2.49 0.40 3.88 3.84 10.15 NM_024344 NM_173087 NM_173088 NM_173089 NM_173090 NM_212464 NM_212465 NM_212467 218282_at NM_018217 46.40 78.77 1.70 0.23 2.78 0.52 9.79 218327_s_at NM_004782 52.94 128.96 2.44 0.39 5.00 3.26 9.20 202946_s_at NM_014962 27.21 59.36 2.18 0.34 2.50 1.58 7.87 NM_181443 203064_s_at NM_004514 124.55 50.66 2.46 0.39 4.95 1.00 12.43 NM_181430 NM_181431 208896_at NM_006773 114.16 46.90 2.43 0.39 4.71 2.17 9.77 205176_s_at NM_014288 110.04 58.77 1.87 0.27 4.05 1.88 8.65 213761_at NM_017440 97.62 43.75 2.23 0.35 6.15 1.37 7.17 NM_020128 209054_s_at NM_007331 103.36 58.15 1.78 0.25 3.70 2.78 6.97 NM_014919 NM_133330 NM_133331 NM_133332 NM_133333 NM_133334 NM_133335 NM_133336 fold change 3-4.5 212467_at NM_173823 40.63 125.08 3.08 0.49 0.69 3.21 21.68 205128_x_at NM_000962 58.26 249.54 4.28 0.63 11.31 2.21 14.14 NM_080591 214544_s_at NM_003825 43.98 136.04 3.09 0.49 6.06 1.59 12.03 NM_130798 217783_s_at NM_016061 51.52 214.96 4.17 0.62 6.70 7.03 11.90 204417_at NM_000153 46.08 163.45 3.55 0.55 4.18 7.57 9.98 202557_at NM_006948 113.75 30.10 3.78 0.58 5.27 1.27 12.79 208433_s_at NM_004631 168.09 49.49 3.40 0.53 9.79 3.58 8.87 NM_017522 NM_033300 203362_s_at NM_002358 218.12 52.85 4.13 0.62 15.89 3.67 8.45 208962_s_at NM_013402 165.07 37.06 4.45 0.65 8.70 7.42 7.94 203627_at NM_000875 111.98 35.96 3.11 0.49 6.82 3.90 7.09 NM_015883 fold change >5 207111_at NM_001974 39.97 287.27 7.19 0.86 2.28 4.89 34.51 205786_s_at NM_000632 51.38 331.91 6.46 0.81 7.15 4.53 24.01 212412_at NM_006457 47.38 242.16 5.11 0.71 6.38 4.85 17.34 204446_s_at NM_000698 50.70 563.72 11.12 1.05 5.18 26.90 15.99 210724_at NM_032571 26.85 278.89 10.39 1.02 1.98 17.05 13.24 NM_152939 210254_at NM_006138 500.13 43.80 11.42 1.06 11.55 3.22 30.90 212563_at NM_015201 189.55 30.71 6.17 0.79 1.90 3.97 27.08 204538_x_at NM_006985 298.36 28.02 10.65 1.03 12.03 4.11 16.76 221539_at NM_004095 622.12 51.77 12.02 1.08 18.14 20.13 14.90 222036_s_at NM_005914 243.17 44.11 5.51 0.74 18.70 5.26 8.31 NM_182746 basal expression level 60-125 units fold change 1.5-2.5 201779_s_at NM_007282 121.10 297.22 2.45 0.39 2.64 11.71 12.27 NM_183381 NM_183382 NM_183383 NM_183384 211067_s_at NM_003644 122.85 267.79 2.18 0.34 8.26 5.49 10.54 NM_005890 NM_201432 NM_201433 202923_s_at NM_001498 63.33 145.68 2.30 0.36 4.04 4.23 9.96 204295_at NM_003172 123.97 211.17 1.70 0.23 5.99 3.85 8.86 207629_s_at NM_004723 103.61 177.50 1.71 0.23 5.56 2.82 8.82 217850_at NM_014366 291.05 119.42 2.44 0.39 2.98 4.54 22.82 NM_206825 NM_206826 203315_at NM_001004720 121.02 61.68 1.96 0.29 0.66 2.06 21.78 NM_001004722 NM_003581 218607_s_at NM_018115 160.90 96.30 1.67 0.22 1.92 4.54 9.99 209511_at NM_021974 127.46 83.32 1.53 0.18 2.55 1.92 9.87 221699_s_at NM_024045 189.21 93.24 2.03 0.31 4.49 5.34 9.77 fold change 3-4.5 202902_s_at NM_004079 65.75 262.67 3.99 0.60 8.96 3.98 15.22 201413_at NM_000414 77.30 335.21 4.34 0.64 10.18 8.52 13.79 212135_s_at NM_001001396 92.80 332.51 3.58 0.55 2.52 14.99 13.69 NM_001684 208485_x_at NM_003879 60.99 214.30 3.51 0.55 7.62 5.12 12.04 201565_s_at NM_002166 105.04 340.67 3.24 0.51 6.80 12.79 12.03 208581_x_at NM_005952 305.95 93.48 3.27 0.51 10.39 2.12 16.98 201890_at NM_001034 352.52 104.62 3.37 0.53 13.89 2.55 15.08 201516_at NM_003132 428.63 113.75 3.77 0.58 19.76 2.03 14.45 221652_s_at NM_018164 280.86 78.45 3.58 0.55 13.83 3.01 12.02 212282_at NM_014573 300.99 96.70 3.11 0.49 11.04 8.12 10.66 fold change >5 209030_s_at NM_014333 114.63 3138.68 27.38 1.44 8.58 21.28 101.28 200701_at NM_006432 101.26 992.64 9.80 0.99 5.45 8.88 62.17 209949_at NM_000433 64.04 431.32 6.74 0.83 5.21 3.41 42.63 202838_at NM_000147 98.39 1727.68 17.56 1.24 17.24 66.39 19.48 211506_s_at NM_000584 91.45 598.35 6.54 0.82 4.81 24.33 17.40 201013_s_at NM_006452 645.25 105.67 6.11 0.79 2.52 4.11 81.40 201930_at NM_005915 633.11 107.33 5.90 0.77 4.02 10.80 35.48 204351_at NM_005980 1257.67 72.27 17.40 1.24 36.81 20.07 20.84 200790_at NM_002539 949.56 101.20 9.38 0.97 63.91 4.53 12.40 202887_s_at NM_019058 508.55 89.10 5.71 0.76 31.95 14.40 9.05 basal expression level >125 units fold change 1.5-2.5 200077_s_at NM_004152 2228.65 3478.72 1.56 0.19 36.65 7.31 28.43 207320_x_at NM_004602 159.09 243.61 1.53 0.19 4.33 0.65 16.96 NM_017452 NM_017453 NM_017454 208641_s_at NM_006908 125.43 286.94 2.29 0.36 1.61 7.94 16.91 NM_018890 NM_198829 213867_x_at NM_001101 6437.29 10848.75 1.69 0.23 107.58 169.49 15.92 204158_s_at NM_006019 183.26 446.89 2.44 0.39 3.84 12.91 15.74 NM_006053 200691_s_at NM_004134 450.19 188.06 2.39 0.38 10.10 6.16 16.12 201077_s_at NM_001003796 675.17 379.69 1.78 0.25 11.15 7.98 15.45 NM_005008 217810_x_at NM_020117 352.53 218.24 1.62 0.21 5.20 3.67 15.14 200792_at NM_001469 940.53 580.29 1.62 0.21 23.54 5.17 12.55 218140_x_at NM_021203 400.95 197.86 2.03 0.31 8.19 8.61 12.09 fold change 3-4.5 210908_s_at NM_002624 857.33 2675.14 3.12 0.49 20.67 51.57 25.16 NM_145896 NM_145897 201460_at NM_004759 142.58 473.41 3.32 0.52 4.71 9.73 22.92 NM_032960 203470_s_at NM_002664 167.89 689.86 4.11 0.61 3.62 23.36 19.34 202803_s_at NM_000211 558.85 2149.86 3.85 0.59 30.29 61.10 17.41 209124_at NM_002468 168.56 687.89 4.08 0.61 7.63 22.94 16.99 201892_s_at NM_000884 1690.72 556.27 3.04 0.48 43.73 15.45 19.17 200647_x_at NM_003752 2203.38 717.78 3.07 0.49 84.31 29.06 13.10 218512_at NM_018256 458.15 145.51 3.15 0.50 13.13 10.86 13.03 209932_s_at NM_001948 783.00 248.26 3.15 0.50 15.57 29.24 11.93 200650_s_at NM_005566 1944.97 593.69 3.28 0.52 90.23 31.23 11.13 fold change >5 217733_s_at NM_021103 637.96 3221.75 5.05 0.70 33.65 82.85 22.18 210592_s_at NM_002970 157.29 1070.71 6.81 0.83 11.56 37.71 18.54 204122_at NM_003332 456.11 3465.79 7.60 0.88 14.27 154.50 17.83 NM_198125 204232_at NM_004106 200.54 1713.24 8.54 0.93 14.01 80.44 16.02 216598_s_at NM_002982 132.79 5147.99 38.77 1.59 27.61 322.89 14.31 204798_at NM_005375 877.47 132.27 6.63 0.82 20.74 14.06 21.41 203949_at NM_000250 2732.30 170.06 16.07 1.21 148.73 13.39 15.80 202107_s_at NM_004526 696.44 137.07 5.08 0.71 48.08 4.62 10.61 211951_at NM_004741 752.52 135.10 5.57 0.75 42.57 19.86 9.89 202431_s_at NM_002467 2723.42 174.53 15.60 1.19 381.41 6.76 6.57

TABLE 3 Probe Sequences signature genes: Flex Affymetrix MAP downstream probe ID RefSeq ID RefSet ID ID upstream probe sequence sequence 200721_s_at NM_005736 HG_010_01195 LUA#1 TAATACGACTCACTATAGGGCTTT seq 1 CCCAGTGTACTGAA seq 91 AATCTCAATCAATACAAATCAACC id ATAAAGTCCCTTTA id ACATTGCCTGGTGGGG no: GTGAGGGTTAAT no: 210944_s_at NM_000070 HG_010_18277 LUA#2 TAATACGACTCACTATAGGGCTTT seq 2 GACGCAGGATTCCA seq 92 ATCAATACATACTACAATCAAGAT id CCTCAATCCCTTTA id GCGAAATGCAGTCAAC no: GTGAGGGTTAAT no: 218282_at NM_018217 HG_010_21926 LUA#3 TAATACGACTCACTATAGGGTACA seq 3 CATTACTGGGACAG seq 93 CTTTATCAAATCTTACAATCGCCC id GTTTTCTCCCTTTA id TTCACCTCCAAGTTGG no: GTGAGGGTTAAT no: 218327_s_at NM_004782 HG_010_06845 LUA#4 TAATACGACTCACTATAGGGTACA seq 4 GGTTCCACTTACTG seq 94 TTACCAATAATCTTCAAATCGCAG id TAATTGTCCCTTTA id AGCAGCTTTTGTGCAC no: GTGAGGGTTAAT no: 202946_s_at NM_014962 HG_010_21147 LUA#5 TAATACGACTCACTATAGGGCAAT seq 5 GTTGTTCATTCTGG seq 95 TCAAATCACAATAATCAATCTCTG id GGATAATCCCTTTA id GCTGGCAGTCTTTGTC no: GTGAGGGTTAAT no: 203064_s_at NM_004514 HG_010_18737 LUA#46 TAATACGACTCACTATAGGGTACA seq 6 CATGTGGCTCGCGT seq 96 TCAACAATTCATTCAATACATTTA id GGACAGTCCCTTTA id TCCACCTCCATTTCAG no: GTGAGGGTTAAT no: 208896_at NM_006773 HG_010_01959 LUA#47 TAATACGACTCACTATAGGGCTTC seq 7 CTGTGCTCACTGCT seq 97 TCATTAACTTACTTCATAATGATT id GTAAAATCCCTTTA id TTTGTGGCATGGATTG no: GTGAGGGTTAAT no: 205176_s_at NM_014288 HG_010_08052 LUA#48 TAATACGACTCACTATAGGGAAAC seq 8 CACTCACCATGAGC seq 98 AAACTTCACATCTCAATAATTGAG id ACCAACTCCCTTTA id GCATTAAGAAGAAATG no: GTGAGGGTTAAT no: 213761_at NM_017440 HG_010_16616 LUA#49 TAATACGACTCACTATAGGGTCAT seq 9 CAGAACCAGAAGCC seq 99 CAATCTTTCAATTTACTTACGAGC id  CCGGAATCCCTTTA id AATGTGGTTGCATCAC no:  GTGAGGGTTAAT no: 209054_s_at NM_007331 HG_010_20167 LUA#50 TAATACGACTCACTATAGGGCAAT seq 10 GGCAGCATCTTCAG seq 100 ATACCAATATCATCATTTACAAGC id CTCTTGTCCCTTTA id GAAATCGGGCTTCCAC no: GTGAGGGTTAAT no: 212467_at NM_173823 * LUA#6 TAATACGACTCACTATAGGGTCAA seq 11 CTGCCACCTCCTGT seq 101 CAATCTTTTACAATCAAATCCTAC id AGACCATCCCTTTA id ATCAGTCATGTCTAAC no: GTGAGGGTTAAT no: 205128_x_at NM_000962 HG_010_04807 LUA#7 TAATACGACTCACTATAGGGCAAT seq 12 CCTGCTAGTCTGCC seq 102 TCATTTACCAATTTACCAATACTC id CTATGGTCCCTTTA id CTGCCTGAGTTTCCAG no: GTGAGGGTTAAT no: 214544_s_at NM_003825 HG_010_06841 LUA#8 TAATACGACTCACTATAGGGAATC seq 13 CATAATCAAGTTGA seq 103 CTTTTACATTCATTACTTACCTTG id TGTGGATCCCTTTA id TGTATTGAACTATGTC no: GTGAGGGTTAAT no: 217783_s_at NM_016061 HG_010_21524 LUA#9 TAATACGACTCACTATAGGGTAAT seq 14 CTATTTGCCACTGG seq 104 CTTCTATATCAACATCTTACTGAG id GCTGTTTCCCTTTA id TACAGTTAAGTTCCTC no: GTGAGGGTTAAT no: 204417_at NM_000153 HG_010_18368 LUA#10 TAATACGACTCACTATAGGGATCA seq 15 CTCAGTCAGTTCCT seq 105 TACATACATACAAATCTACAAAGG id TTCACTTCCCTTTA id TTCTCTTGTATACCTG no: GTGAGGGTTAAT no: 202557_at NM_006948 HG_010_16269 LUA#51 TAATACGACTCACTATAGGGTCAT seq 16 CTCATCTCATGTCC seq 106 TTCAATCAATCATCAACAATTGAC id TGAAGTTCCCTTTA id AAAATAGGGCAGGCAG no: GTGAGGGTTAAT no: 208433_s_at NM_004631 HG_010_03370 LUA#52 TAATACGACTCACTATAGGGTCAA seq 17 CTGGAGAACGAGGC seq 107 TCATCTTTATACTTCACAATACAA id CATTTTTCCCTTTA id GGTGTTCTGGACAGAC no: GTGAGGGTTAAT no: 203362_s_at NM_002358 HG_010_20134 LUA#53 TAATACGACTCACTATAGGGTAAT seq 18 GTCAAGTAGTTTGA seq 108 TATACATCTCATCTTCTACATTCC id CTCAGTTCCCTTTA id TAAATCAGATGTTTTG no: GTGAGGGTTAAT no: 208962_s_at NM_013402 HG_010_02173 LUA#54 TAATACGACTCACTATAGGGCTTT seq 19 CCTTCTCAGCCTAC seq 109 TTCAATCACTTTCAATTCATAAGC id AGCAGTTCCCTTTA id ACCTGAACCACTGTGG no: GTGAGGGTTAAT no: 203627_at NM_000875 HG_010_00403 LUA#55 TAATACGACTCACTATAGGGTATA seq 20 CTTCTGACTAGATT seq 110 TACACTTCTCAATAACTAACCAGG id ATTATTTCCCTTTA id CACACAGGTCTCATTG no: GTGAGGGTTAAT no: 207111_at NM_001974 HG_010_17076 LUA#11 TAATACGACTCACTATAGGGTACA seq 21 CACTGATGAGAAAT seq 111 AATCATCAATCACTTTAATCCGTC id CAGACGTCCCTTTA id TTCCTGTGGTTGTATG no: GTGAGGGTTAAT no: 205786_s_at NM_000632 HG_010_20041 LUA#12 TAATACGACTCACTATAGGGTACA seq 22 CAGGCGATGTGCAA seq 112 CTTTCTTTCTTTCTTTCTTTGGTT id GTGTATTCCCTTTA id TCCTTCAGACAGATTC no: GTGAGGGTTAAT no: 212412_at NM_006457 HG_010_19532 LUA#13 TAATACGACTCACTATAGGGCAAT seq 23 GATCAGTGGCACCA seq 113 AAACTATACTTCTTCACTAAAAAC id GCCAACTCCCTTTA id AGCGCTACTTACTCAG no: GTGAGGGTTAAT no: 204446_s_at NM_000698 HG_010_16744 LUA#14 TAATACGACTCACTATAGGGCTAC seq 24 CAGCAACAGCAAAT seq 114 TATACATCTTACTATACTTTCTCA id CACGACTCCCTTTA id GCATTTCCACACCAAG no: GTGAGGGTTAAT no: 210724_at NM_032571 HG_010_15648 LUA#15 TAATACGACTCACTATAGGGATAC seq 25 CTGACTCAAAACCC seq 115 TTCATTCATTCATCAATTCAACTT id AGTGAGTCCCTTTA id TCCAGCAAGATGGGTC no: GTGAGGGTTAAT no: 210254_at NM_006138 HG_010_15460 LUA#56 TAATACGACTCACTATAGGGCAAT seq 26 GAACTCACACATGC seq 116 TTACTCATATACATCACTTTTTTA id CCTGATTCCCTTTA id TTTCAGTGAACTGCTG no: GTGAGGGTTAAT no: 212563_at NM_015201 HG_010_10972 LUA#57 TAATACGACTCACTATAGGGCAAT seq 27 CTGGTGTGGTTTGA seq 117 ATCATCATCTTTATCATTACGTGG id CCTGGATCCCTTTA id GAGCTACGATAGCAAG no: GTGAGGGTTAAT no: 204538_x_at NM_006985 * LUA#58 TAATACGACTCACTATAGGGCTAC seq 28 GGAGTGTCTGCTCT seq 118 TAATTCATTAACATTACTACGATA id ATCCCCTCCCTTTA id ATCTCAAGACACCTGC no: GTGAGGGTTAAT no: 221539_at NM_004095 HG_010_07678 LUA#59 TAATACGACTCACTATAGGGTCAT seq 29 GGAAAGCTCCCTCC seq 119 CAATCAATCTTTTTCACTTTTCCT id CCCTCCTCCCTTTA id TAGGTTGATGTGCTTG no: GTGAGGGTTAAT no: 222036_s_at NM_005914 * LUA#60 TAATACGACTCACTATAGGGAATC seq 30 GCTTAAACCCAGGC seq 120 TACAAATCCAATAATCTCATGAGG id GGCAGATCCCTTTA id TTGAGGCAGGAGAATC no: GTGAGGGTTAAT no: 201779_s_at NM_007282 HG_010_08042 LUA#16 TAATACGACTCACTATAGGGAATC  seq 31 GAGAGGCAACAAGG seq 121 AATCTTCATTCAAATCATCACTGA id TAATTCTCCCTTTA id CCTGCCAATCATTAGG no: GTGAGGGTTAAT no: 211067_s_at NM_003644 HG_010_17163 LUA#17 TAATACGACTCACTATAGGGCTTT seq 32 GAGAATCAGACAGA seq 122 AATCCTTTATCACTTTATCACCAT id GGGCAATCCCTTTA id TGCAGCAGGTTAGAGC no: GTGAGGGTTAAT no: 202923_s_at NM_001498 HG_010_18372 LUA#18 TAATACGACTCACTATAGGGTCAA seq 33 CCCCAAGCTTTCCC seq 123 AATCTCAAATACTCAAATCAATAA id CTCTGATCCCTTTA id TCACTTGGTCACCTTG no: GTGAGGGTTAAT no: 204295_at NM_003172 HG_010_06973 LUA#19 TAATACGACTCACTATAGGGTCAA seq 34 CATTATCGAGACCT seq 124 TCAATTACTTACTCAAATACATCC id GGAAGCTCCCTTTA id AGAAAGGAACCACTGG no: GTGAGGGTTAAT no: 207629_s_at NM_004723 HG_010_03179 LUA#20 TAATACGACTCACTATAGGGCTTT seq 35 CAACCATGACCTGA seq 125 TACAATACTTCAATACAATCGACC id AACCTCTCCCTTTA id TCATCTTCCACCTCAG no: GTGAGGGTTAAT no: 217850_at NM_014366 HG_010_20659 LUA#61 TAATACGACTCACTATAGGGAATC seq 36 CAGGTGAACAGTCT seq 126 TTACCAATTCATAATCTTCACACT id ACAAGGTCCCTTTA id TCTGAGGAGACTACAG no: GTGAGGGTTAAT no: 203315_at NM_003581 HG_010_17522 LUA#62 TAATACGACTCACTATAGGGTCAA seq 37 GTCAGGGAAGAACA seq 127 TCATAATCTCATAATCCAATTTCT id AACACTTCCCTTTA id CCGTGTCCCTTAAAGC no: GTGAGGGTTAAT no: 218607_s_at NM_018115 HG_010_21859 LUA#63 TAATACGACTCACTATAGGGCTAC seq 38 CCTGTAATATTTTC seq 128 TTCATATACTTTATACTACATTTC id AGCCCATCCCTTTA id CTCAGCCTTCCTTCAG no: GTGAGGGTTAAT no: 209511_at NM_021974 HG_010_02843 LUA#64 TAATACGACTCACTATAGGGCTAC seq 39 GAGTCATCTTCCTG seq 129 ATATTCAAATTACTACTTACCATC id CCCTTGTCCCTTTA id ATCACCGACTGAGCTG no: GTGAGGGTTAAT no: 221699_s_at NM_024045 HG_010_01029 LUA#65 TAATACGACTCACTATAGGGCTTT seq 40 CATCAAGCTTTGAA seq 130 TCATCAATAATCTTACCTTTTTTA id CCACGATCCCTTTA id GCCCACATTTCTGGTG no: GTGAGGGTTAAT no: 202902_s_at NM_004079 HG_010_15445 LUA#21 TAATACGACTCACTATAGGGAATC seq 41 GAATCTAAACAAAC seq 131 CTTTCTTTAATCTCAAATCAAAGC id AGGCCTTCCCTTTA id ACAGGGACACAAAGAG no: GTGAGGGTTAAT no: 201413_at NM_000414 HG_010_17294 LUA#22 TAATACGACTCACTATAGGGAATC seq 42 CCAGAGGGAACATC seq 132 CTTTTTACTCAATTCAATCACTTT id ATGCTGTCCCTTTA id AGTGGCAGGCTGAAGG no: GTGAGGGTTAAT no: 212135_s_at NM_001684 HG_010_16788 LUA#23 TAATACGACTCACTATAGGGTTCA seq 43 CATCACCCCACCCC seq 133 ATCATTCAAATCTCAACTTTAATG id ACATTCTCCCTTTA id ATGACAATCCTCTTGG no: GTGAGGGTTAAT no: 208485_x_at NM_003879 * LUA#24 TAATACGACTCACTATAGGGTCAA seq 44 CACACTCTGAGAAA seq 134 TTACCTTTTCAATACAATACAATA id GAAACTTCCCTTTA id TTATGTCTGGCTGCAG no: GTGAGGGTTAAT no: 201565_s_at NM_002166 HG_010_17313 LUA#25 TAATACGACTCACTATAGGGCTTT seq 45 CCTTCTGAGTTAAT seq 135 TCAATTACTTCAAATCTTCACCTT id GTCAAATCCCTTTA id GCAGGCTTCTGAATTC no: GTGAGGGTTAAT no: 208581_x_at NM_005952 * LUA#66 TAATACGACTCACTATAGGGTAAC seq 46 CAACCTATATAAAC seq 136 ATTACAACTATACTATCTACGCTC id CTGGATTCCCTTTA id TCAGATGTAAATAGAG no: GTGAGGGTTAAT no: 201890_at NM_001034 HG_010_18467 LUA#67 TAATACGACTCACTATAGGGTCAT seq 47 CCCCTCTGAGTAGA seq 137 TTACTCAACAATTACAAATCAGTG id GTGTTGTCCCTTTA id TCCTGGGATTCTCTGC no: GTGAGGGTTAAT no: 201516_at NM_003132 HG_010_17983 LUA#68 TAATACGACTCACTATAGGGTCAT seq 48 CCTATACCAGCTGT seq 138 AATCTCAACAATCTTTCTTTTCTG id GTACAGTCCCTTTA id GCGTTCCACCTCCAAG no: GTGAGGGTTAAT no: 221652_s_at NM_018164 HG_010_00331 LUA#69 TAATACGACTCACTATAGGGCTAT seq 49 GGCAGTGAAGAGTG seq 139 AAACATATTACATTCACATCAGAA id ACTTGATCCCTTTA id AATGGAAAAGCCAGCC no: GTGAGGGTTAAT no: 212282_at NM_014573 * LUA#70 TAATACGACTCACTATAGGGATAC seq 50 CATCTCAAGGCTGA seq 140 CAATAATCCAATTCATATCATCCC id TCTGGATCCCTTTA id TGTATCTGAAGTCTAG no: GTGAGGGTTAAT no: 209030_s_at NM_014333 HG_010_14934 LUA#26 TAATACGACTCACTATAGGGTTAC seq 51 GCACTTAACCAAGA seq 141 TCAAAATCTACACTTTTTCATACC id CAAAAATCCCTTTA id CCTCCCCTATCCCTAG no: GTGAGGGTTAAT no: 200701_at NM_006432 HG_010_08035 LUA#27 TAATACGACTCACTATAGGGCTTT seq 52 GCTGGTTCTCAGTG seq 142 TCAAATCAATACTCAACTTTCAGA id GTTGTCTCCCTTTA id AACTGAGCTCCGGGTG no: GTGAGGGTTAAT no: 209949_at NM_000433 HG_010_18441 LUA#28 TAATACGACTCACTATAGGGCTAC seq 53 CAGGTACTGATCCT seq 143 AAACAAACAAACATTATCAAAAGG id GTTTCTTCCCTTTA id GCACGAGAGAGTCTTC no: GTGAGGGTTAAT no: 202838_at NM_000147 HG_010_16435 LUA#29 TAATACGACTCACTATAGGGAATC seq 54 CTATGGTCAACTCT seq 144 TTACTACAAATCCTTTCTTTGGAA id TCAGAATCCCTTTA id AAGGCTTACCAGGCTG no: GTGAGGGTTAAT no: 211506_s_at NM_000584 HG_010_00131 LUA#30 TAATACGACTCACTATAGGGTTAC seq 55 CAGTCTTGTCATTG seq 145 CTTTATACCTTTCTTTTTACCAAT id CCAGCTTCCCTTTA id CCTAGTTTGATACTCC no: GTGAGGGTTAAT no: 201013_s_at NM_006452 HG_010_04110 LUA#71 TAATACGACTCACTATAGGGATCA seq 56 CTTTAGTTCTCTGA seq 146 TTACAATCCAATCAATTCATGGAC id AGGCCCTCCCTTTA id TGCCACACATTGGTAC no: GTGAGGGTTAAT no: 201930_at NM_005915 HG_010_16268 LUA#72 TAATACGACTCACTATAGGGTCAT seq 57 CCTTGATGTCTGAG seq 147 TTACCTTTAATCCAATAATCACCC id CTTTCCTCCCTTTA id ATGAGTACTCAACTTG no: GTGAGGGTTAAT no: 204351_at NM_005980 HG_010_19452 LUA#73 TAATACGACTCACTATAGGGATCA seq 58 CCGTGGATAAATTG seq 148 AATCTCATCAATTCAACAATGAGT id CTCAAGTCCCTTTA id GGAAAAGACAAGGATG no: GTGAGGGTTAAT no: 200790_at NM_002539 HG_010_17575 LUA#74 TAATACGACTCACTATAGGGTACA seq 59 CATTTGTAGCTTGT seq 149 CATCTTACAAACTAATTTCACCCC id ACAATGTCCCTTTA id TCAGCTGCTGAACAAG no: GTGAGGGTTAAT no: 202887_s_at NM_019058 * LUA#75 TAATACGACTCACTATAGGGAATC seq 60 CCTTCCCCCATCGT seq 150 ATACCTTTCAATCTTTTACAACCT id GTACTGTCCCTTTA id GGCAGCTGCGTTTAAG no: GTGAGGGTTAAT no: 200077_s_at NM_004152 HG_010_22476 LUA#31 TAATACGACTCACTATAGGGTTCA seq 61 GTGCAAATAAACGC seq 151 CTTTTCAATCAACTTTAATCTTTG id TCACTCTCCCTTTA id TCCGCATGTTGTAATC no: GTGAGGGTTAAT no: 207320_x_at NM_004602 HG_010_18893 LUA#32 TAATACGACTCACTATAGGGATTA seq 62 AGAACTAAATGCAC seq 152 TTCACTTCAAACTAATCTACGAAA id TGTGCATCCCTTTA id GCATAACCCCTACTGT no: GTGAGGGTTAAT no: 208641_s_at NM_018890 HG_010_22573 LUA#33 TAATACGACTCACTATAGGGTCAA seq 63 GAGAAGAAGCTGAC seq 153 TTACTTCACTTTAATCCTTTACAC id TCCCATTCCCTTTA id GATCGAGAAACTGAAG no: GTGAGGGTTAAT no: 213867_x_at NM_001101 HG_010_19208 LUA#34 TAATACGACTCACTATAGGGTCAT seq 64 CACACAGGGGAGGT seq 154 TCATATACATACCAATTCATGCCC id GATAGCTCCCTTTA id AGTCCTCTCCCAAGTC no: GTGAGGGTTAAT no: 204158_s_at NM_006019 HG_010_07626 LUA#35 TAATACGACTCACTATAGGGCAAT seq 65 GCATCTGTGAATGG seq 155 TTCATCATTCATTCATTTCAGGTT id CTGGAGTCCCTTTA id GCTGGACCTGCCTGAC no: GTGAGGGTTAAT no: 200691_s_at NM_004134 HG_010_15879 LUA#76 TAATACGACTCACTATAGGGAATC seq 66 CTGTGTCTGGCACC seq 156 TAACAAACTCATCTAAATACTTTT id TACATCTCCCTTTA id CTAGCTACCTTCTGCC no: GTGAGGGTTAAT no: 201077_s_at NM_005008 HG_010_18994 LUA#77 TAATACGACTCACTATAGGGCAAT seq 67 CTGGCATGAAGGAT seq 157 TAACTACATACAATACATACTCAG id TCCAGGTCCCTTTA id AGAGCATGAACTGATG no: GTGAGGGTTAAT no 217810_x_at NM_020117 HG_010_16506 LUA#78 TAATACGACTCACTATAGGGCTAT seq 68 GCTATCAGAACCTT seq 158 CTATCTAACTATCTATATCACTGA id AGGCTGTCCCTTTA id TTGTGTCTACTGATTG no: GTGAGGGTTAAT no: 200792_at NM_001469 HG_010_07661 LUA#79 TAATACGACTCACTATAGGGTTCA seq 69 GTGTAGCCCTCCCA seq 159 TAACTACAATACATCATCATTTTC id CTTTGCTCCCTTTA id TGTTGCCATGGTGATG no: GTGAGGGTTAAT no: 218140_x_at NM_021203 HG_010_03138 LUA#80 TAATACGACTCACTATAGGGCTAA seq 70 CTGCTCTGCTGCTC seq 160 CTAACAATAATCTAACTAACAGTG id TGGATGTCCCTTTA id TGTGGAGATTTAGGTG no: GTGAGGGTTAAT no: 210908_s_at NM_002624 HG_010_15000 LUA#36 TAATACGACTCACTATAGGGCAAT seq 71 GAGAAGCACGCCAT seq 161 TCATTTCATTCACAATCAATAAAT id GAAACATCCCTTTA id CCAACCAGCTCTTCAG no: GTGAGGGTTAAT no: 201460_at NM_004759 HG_010_02788 LUA#37 TAATACGACTCACTATAGGGCTTT seq 72 CAATAACTCTCTAC seq 162 TCATCTTTTCATCTTTCAATCCTG id AGGAATTCCCTTTA id CCCACGGGAGGACAAG no: GTGAGGGTTAAT no: 203470_s_at NM_002664 HG_010_17685 LUA#38 TAATACGACTCACTATAGGGTCAA seq 73 CTGTTCCCACTCCC seq 163 TCATTACACTTTTCAACAATCCCC id AGATGGTCCCTTTA id TGTAACATTCCTGAAG no: GTGAGGGTTAAT no: 202803_s_at NM_000211 HG_010_18487 LUA#39 TAATACGACTCACTATAGGGTACA seq 74 CCCTCAAAATGACA seq 164 CAATCTTTTCATTACATCATAGAA id GCCATGTCCCTTTA id ATCCAGTTATTTTCCG no: GTGAGGGTTAAT no: 209124_at NM_002468 HG_010_07210 LUA#40 TAATACGACTCACTATAGGGCTTT seq 75 CCATGCACCTGTCC seq 165 CTACATTATTCACAACATTACTTG id CCCTTTTCCCTTTA id TTGAGGCATTTAGCTG no: GTGAGGGTTAAT no: 201892_s_at NM_000884 HG_010_17352 LUA#81 TAATACGACTCACTATAGGGCTTT seq 76 CTGGCATCCAACAC seq 166 AATCTACACTTTCTAACAATATTT id TCATGCTCCCTTTA id GTCCCTTACCTGATTG no: GTGAGGGTTAAT no: 200647_x_at NM_003752 HG_010_19669 LUA#82 TAATACGACTCACTATAGGGTACA seq 77 CTGCTACCACATGA seq 167 TACACTAATAACATACTCATTTGC id CAGACATCCCTTTA id TGATTATACTTCTGAG no: GTGAGGGTTAAT no: 218512_at NM_018256 HG_010_03754 LUA#83 TAATACGACTCACTATAGGGATAC seq 78 GACAGACACAGGGC seq 168 AATCTAACTTCACTATTACAAAAG id TACTTCTCCCTTTA id TTCTGAGTGTAGACTG no: GTGAGGGTTAAT no: 209932_s_at NM_001948 HG_010_10582 LUA#84 TAATACGACTCACTATAGGGTCAA seq 79 CACAGGCAAGAGTG seq 169 CTAACTAATCATCTATCAATGACC id TTCTTTTCCCTTTA id ACCCAGTTTGTGGAAG no: GTGAGGGTTAAT no: 200650_s_at NM_005566 HG_010_19291 LUA#85 TAATACGACTCACTATAGGGATAC seq 80 GCACCACTGCCAAT seq 170 TACATCATAATCAAACATCAATAG id GCTGTATCCCTTTA id TTCTGCCACCTCTGAC no: GTGAGGGTTAAT no: 217733_s_at NM_021103 HG_010_00217 LUA#41 TAATACGACTCACTATAGGGTTAC seq 81 GAGAAGCGGAGTGA seq 171 TACACAATATACTCATCAATCCAA id AATTTCTCCCTTTA id AGAGACCATTGAGCAG no: GTGAGGGTTAAT no: 210592_s_at NM_002970 HG_010_17875 LUA#42 TAATACGACTCACTATAGGGCTAT seq 82 GAGTGCTGCTGTAG seq 172 CTTCATATTTCACTATAAACAATG id ATGACATCCCTTTA id GCAACAGAGGAGTGAG no: GTGAGGGTTAAT no: 204122_at NM_003332 HG_010_18121 LUA#43 TAATACGACTCACTATAGGGCTTT seq 83 CAGACCGCTCCCCA seq 173 CAATTACAATACTCATTACAGAGT id ATACTCTCCCTTTA id GCCATCCCTGAGAGAC no: GTGAGGGTTAAT no: 204232_at NM_004106 HG_010_18680 LUA#44 TAATACGACTCACTATAGGGTCAT seq 84 GAGACTCTGAAGCA seq 174 TTACCAATCTTTCTTTATACCCAG id TGAGAATCCCTTTA id GAACCAGGAGACTTAC no: GTGAGGGTTAAT no: 216598_s_at NM_002982 HG_010_15183 LUA#45 TAATACGACTCACTATAGGGTCAT seq 85 CCTGGGATGTTTTG seq 175 TTCACAATTCAATTACTCAATCTT id AGGGTCTCCCTTTA id GAACCACAGTTCTACC no: GTGAGGGTTAAT no: 204798_at NM_005375 HG_010_19159 LUA#86 TAATACGACTCACTATAGGGCTAA seq 86 CATGGATCCTGTGT seq 176 TTACTAACATCACTAACAATGTAT id TTGCAATCCCTTTA id GGTCTCAGAACTGTTG no: GTGAGGGTTAAT no: 203949_at NM_000250 HG_010_18429 LUA#87 TAATACGACTCACTATAGGGAAAC seq 87 CTTATTCACTGAAG seq 177 TAACATCAATACTTACATCATTCC id TTCTCCTCCCTTTA id TCACCCTGATTTCTTG no: GTGAGGGTTAAT no: 202107_s_at NM_004526 HG_010_18766 LUA#88 TAATACGACTCACTATAGGGTTAC seq 88 CTCCCTGTCTGTTT seq 178 TTCACTTTCTATTTACAATCACAG id CCCCACTCCCTTTA id TTATCAGCTGCCATTG no: GTGAGGGTTAAT no: 211951_at NM_004741 HG_010_18809 LUA#89 TAATACGACTCACTATAGGGTATA seq 89 GGTCTTGATGAGGA seq 179 CTATCAACTCAACAACATATCCCT id CAGAAGTCCCTTTA id CAGGTCTCTAGGTGAG no: GTGAGGGTTAAT no: 202431_s_at NM_002467 HG_010_00920 LUA#90 TAATACGACTCACTATAGGGCTAA seq 90 GTCCAAGCAGAGGA seq 180 ATACTTCACAATTCATCTAACCAC id GCAAAATCCCTTTA id AGCATACATCCTGTCC no: GTGAGGGTTAAT no: control features: FlexMAP description RefSeq ID RefSet ID ID upstream probe sequence downstream probe sequence ACTB NM_001101 * LUA#91 TAATACGACTCACTATAGGGTTCA seq 181 CATTGTTACAGGAA seq 186 TAACATCAATCATAACTTACGTCA id GTCCCTTCCCTTTA id TTCCAAATATGAGATG no: GTGAGGGTTAAT no: TFRC NM_003234 * LUA#92 TAATACGACTCACTATAGGGCTAT seq 182 GTGATCAATTAAAT seq 187 TACACTTTAAACATCAATACCGTC id GTAGGTTCCCTTTA id TGCCTACCCATTCGTG no: GTGAGGGTTAAT no: GAPDH_5 NM_002046 * LUA#93 TAATACGACTCACTATAGGGCTTT seq 183 GTTTACATGTTCCA seq 188 CTATTCATCTAAATACAAACTCAT id ATATGATCCCTTTA id TGACCTCAACTACATG no: GTGAGGGTTAAT no: GAPDH_M NM_002046 * LUA#94 TAATACGACTCACTATAGGGCTTT seq 184 CCACCCAGAAGACT seq 189 CTATCTTTCTACTCAATAATCACA id GTGGATTCCCTTTA id GTCCATGCCATCACTG no: GTGAGGGTTAAT no: GAPDH_3 NM_002046 * LUA#95 TAATACGACTCACTATAGGGTACA seq 185 CAAGAGCACAAGAG seq 190 CTTTAAACTTACTACACTAACCCT id GAAGAGTCCCTTTA id GGACCACCAGCCCCAG no: GTGAGGGTTAAT no: * probes designed against RefSeq FlexMAP sequence shown in red gene specific sequences shown in blue FlexMAP sequence of upstream primer bases 21-44 gene specific sequences of upstream probe bases 45-64 gene specific sequences of downstream probe bases 1-20

TABLE 4 Capture Probes FlexMAP bead ID ID capture probe sequence Bead #1 LUA-1 GATTTGTATTGATTGAGATTAAAG seq id no: 191 Bead #2 LUA-2 TGATTGTAGTATGTATTGATAAAG seq id no: 192 Bead #3 LUA-3 GATTGTAAGATTTGATAAAGTGTA seq id no: 193 Bead #4 LUA-4 GATTTGAAGATTATTGGTAATGTA seq id no: 194 Bead #5 LUA-5 GATTGATTATTGTGATTTGAATTG seq id no: 195 Bead #46 LUA-46 TGTATTGAATGAATTGTTGATGTA seq id no: 196 Bead #47 LUA-47 ATTATGAAGTAAGTTAATGAGAAG seq id no: 197 Bead #48 LUA-48 ATTATTGAGATGTGAAGTTTGTTT seq id no: 198 Bead #49 LUA-49 GTAAGTAAATTGAAAGATTGATGA seq id no: 199 Bead #50 LUA-50 GTAAATGATGATATTGGTATATTG seq id no: 200 Bead #6 LUA-6 GATTTGATTGTAAAAGATTGTTGA seq id no: 201 Bead #7 LUA-7 ATTGGTAAATTGGTAAATGAATTG seq id no: 202 Bead #8 LUA-8 GTAAGTAATGAATGTAAAAGGATT seq id no: 203 Bead #9 LUA-9 GTAAGATGTTGATATAGAAGATTA seq id no: 204 Bead #10 LUA-10 TGTAGATTTGTATGTATGTATGAT seq id no: 205 Bead #51 LUA-51 ATTGTTGATGATTGATTGAAATGA seq id no: 206 Bead #52 LUA-52 ATTGTGAAGTATAAAGATGATTGA seq id no: 207 Bead #53 LUA-53 TGTAGAAGATGAGATGTATAATTA seq id no: 208 Bead #54 LUA-54 ATGAATTGAAAGTGATTGAAAAAG seq id no: 209 Bead #55 LUA-55 GTTAGTTATTGAGAAGTGTATATA seq id no: 210 Bead #11 LUA-11 GATTAAAGTGATTGATGATTTGTA seq id no: 211 Bead #12 LUA-12 AAAGAAAGAAAGAAAGAAAGTGTA seq id no: 212 Bead #13 LUA-13 TTAGTGAAGAAGTATAGTTTATTG seq id no: 213 Bead #14 LUA-14 AAAGTATAGTAAGATGTATAGTAG seq id no: 214 Bead #15 LUA-15 TGAATTGATGAATGAATGAAGTAT seq id no: 215 Bead #56 LUA-56 AAAGTGATGTATATGAGTAAATTG seq id no: 216 Bead #57 LUA-57 GTAATGATAAAGATGATGATATTG seq id no: 217 Bead #58 LUA-58 GTAGTAATGTTAATGAATTAGTAG seq id no: 218 Bead #59 LUA-59 AAAGTGAAAAAGATTGATTGATGA seq id no: 219 Bead #60 LUA-60 ATGAGATTATTGGATTTGTAGATT seq id no: 220 Bead #16 LUA-16 TGATGATTTGAATGAAGATTGATT seq id no: 221 Bead #17 LUA-17 TGATAAAGTGATAAAGGATTAAAG seq id no: 222 Bead #18 LUA-18 TGATTTGAGTATTTGAGATTTTGA seq id no: 223 Bead #19 LUA-19 GTATTTGAGTAAGTAATTGATTGA seq id no: 224 Bead #20 LUA-20 GATTGTATTGAAGTATTGTAAAAG seq id no: 225 Bead #61 LUA-61 TGAAGATTATGAATTGGTAAGATT seq id no: 226 Bead #62 LUA-62 ATTGGATTATGAGATTATGATTGA seq id no: 227 Bead #63 LUA-63 TGTAGTATAAAGTATATGAAGTAG seq id no: 228 Bead #64 LUA-64 GTAAGTAGTAATTTGAATATGTAG seq id no: 229 Bead #65 LUA-65 AAAGGTAAGATTATTGATGAAAAG seq id no: 230 Bead #21 LUA-21 TGATTTGAGATTAAAGAAAGGATT seq id no: 231 Bead #22 LUA-22 TGATTGAATTGAGTAAAAAGGATT seq id no: 232 Bead #23 LUA-23 AAAGTTGAGATTTGAATGATTGAA seq id no: 233 Bead #24 LUA-24 GTATTGTATTGAAAAGGTAATTGA seq id no: 234 Bead #25 LUA-25 TGAAGATTTGAAGTAATTGAAAAG seq id no: 235 Bead #66 LUA-66 GTAGATAGTATAGTTGTAATGTTA seq id no: 236 Bead #67 LUA-67 GATTTGTAATTGTTGAGTAAATGA seq id no: 237 Bead #68 LUA-68 AAAGAAAGATTGTTGAGATTATGA seq id no: 238 Bead #69 LUA-69 GATGTGAATGTAATATGTTTATAG seq id no: 239 Bead #70 LUA-70 TGATATGAATTGGATTATTGGTAT seq id no: 240 Bead #26 LUA-26 TGAAAAAGTGTAGATTTTGAGTAA seq id no: 241 Bead #27 LUA-27 AAAGTTGAGTATTGATTTGAAAAG seq id no: 242 Bead #28 LUA-28 TTGATAATGTTTGTTTGTTTGTAG seq id no: 243 Bead #29 LUA-29 AAAGAAAGGATTTGTAGTAAGATT seq id no: 244 Bead #30 LUA-30 GTAAAAAGAAAGGTATAAAGGTAA seq id no: 245 Bead #71 LUA-71 ATGAATTGATTGGATTGTAATGAT seq id no: 246 Bead #72 LUA-72 GATTATTGGATTAAAGGTAAATGA seq id no: 247 Bead #73 LUA-73 ATTGTTGAATTGATGAGATTTGAT seq id no: 248 Bead #74 LUA-74 TGAAATTAGTTTGTAAGATGTGTA seq id no: 249 Bead #75 LUA-75 TGTAAAAGATTGAAAGGTATGATT seq id no: 250 Bead #31 LUA-31 GATTAAAGTTGATTGAAAAGTGAA seq id no: 251 Bead #32 LUA-32 GTAGATTAGTTTGAAGTGAATAAT seq id no: 252 Bead #33 LUA-33 AAAGGATTAAAGTGAAGTAATTGA seq id no: 253 Bead #34 LUA-34 ATGAATTGGTATGTATATGAATGA seq id no: 254 Bead #35 LUA-35 TGAAATGAATGAATGATGAAATTG seq id no: 255 Bead #76 LUA-76 GTATTTAGATGAGTTTGTTAGATT seq id no: 256 Bead #77 LUA-77 GTATGTATTGTATGTAGTTAATTG seq id no: 257 Bead #78 LUA-78 TGATATAGATAGTTAGATAGATAG seq id no: 258 Bead #79 LUA-79 ATGATGATGTATTGTAGTTATGAA seq id no: 259 Bead #80 LUA-80 GTTAGTTAGATTATTGTTAGTTAG seq id no: 260 Bead #36 LUA-36 ATTGATTGTGAATGAAATGAATTG seq id no: 261 Bead #37 LUA-37 ATTGAAAGATGAAAAGATGAAAAG seq id no: 262 Bead #38 LUA-38 ATTGTTGAAAAGTGTAATGATTGA seq id no: 263 Bead #39 LUA-39 ATGATGTAATGAAAAGATTGTGTA seq id no: 264 Bead #40 LUA-40 TAATGTTGTGAATAATGTAGAAAG seq id no: 265 Bead #81 LUA-81 ATTGTTAGAAAGTGTAGATTAAAG seq id no: 266 Bead #82 LUA-82 ATGAGTATGTTATTAGTGTATGTA seq id no: 267 Bead #83 LUA-83 TGTAATAGTGAAGTTAGATTGTAT seq id no: 268 Bead #84 LUA-84 ATTGATAGATGATTAGTTAGTTGA seq id no: 269 Bead #85 LUA-85 TGATGTTTGATTATGATGTAGTAT seq id no: 270 Bead #41 LUA-41 ATTGATGAGTATATTGTGTAGTAA seq id no: 271 Bead #42 LUA-42 GTTTATAGTGAAATATGAAGATAG seq id no: 272 Bead #43 LUA-43 TGTAATGAGTATTGTAATTGAAAG seq id no: 273 Bead #44 LUA-44 GTATAAAGAAAGATTGGTAAATGA seq id no: 274 Bead #45 LUA-45 TTGAGTAATTGAATTGTGAAATGA seq id no: 275 Bead #86 LUA-86 ATTGTTAGTGATGTTAGTAATTAG seq id no: 276 Bead #87 LUA-87 TGATGTAAGTATTGATGTTAGTTT seq id no: 277 Bead #88 LUA-88 GATTGTAAATAGAAAGTGAAGTAA seq id no: 278 Bead #89 LUA-89 ATATGTTGTTGAGTTGATAGTATA seq id no: 279 Bead #90 LUA-90 TTAGATGAATTGTGAAGTATTTAG seq id no: 280 Bead #91 LUA-91 GTAAGTTATGATTGATGTTATGAA seq id no: 281 Bead #92 LUA-92 GTATTGATGTTTAAAGTGTAATAG seq id no: 282 Bead #93 LUA-93 GTTTGTATTTAGATGAATAGAAAG seq id no: 283 Bead #94 LUA-94 ATTATTGAGTAGAAAGATAGAAAG seq id no: 284 Bead #95 LUA-95 TTAGTGTAGTAAGTTTAAAGTGTA seq id no: 285

TABLE 5A-I Microtiter plates Table 5A. Microtiter plates. FlexMap description ID blank blank dmso1 dmso2 dmso3 dmso4 dmso5 dmso6 dmso7 dmso8 dmso9 dmso10 NM_005736 LUA#1 40 33.5 902 774 850.5 914 836.5 900 888 563 803.5 692.5 NM_000070 LUA#2 39 36 653.5 434 571 624 650 609 575.5 265 499.5 499.5 NM_018217 LUA#3 42 30 1547 1243 1382 1463 1448 1444.5 1416 713 1276.5 1180 NM_004782 LUA#4 45 39 1402 1082 1284 1397 1324 1234 1389.5 724.5 1105 1140.5 NM_014962 LUA#5 49 39 1724 1597 1549 1670 1554 1467 1437 732 1251 1222 NM_004514 LUA#46 39 30.5 1490.5 1130 1389 1498 1455 1394 1420.5 804.5 1235 1160.5 NM_006773 LUA#47 34.5 40 682 571 683 734 698 672.5 664 409 683 635 NM_014288 LUA#48 41 37 713 527 655 721 710 761 657 364 672 643 NM_017440 LUA#49 28 32 621 443 568 629 599 613 562 303 499 481 NM_007331 LUA#50 38.5 29 1011 821.5 931.5 956 988 981.5 839 359 755 736 NM_173823 LUA#6 38 27 1411 1222.5 1272.5 1413 1326 1203.5 1333 475 850 861 NM_000962 LUA#7 33 37 472 401 416.5 435 406 368.5 387 138 306 287 NM_003825 LUA#8 42 34.5 574.5 483 474.5 575 482 430.5 434 188 336 324 NM_016061 LUA#9 46 37 1208 1137 1050.5 1049 962 909.5 905 365 714 683 NM_000153 LUA#10 35 43 63 57.5 59 62.5 48 44.5 46 38 46 48 NM_006948 LUA#51 36.5 32.5 71 55 75 68 74 79.5 60.5 46 50 41 NM_004631 LUA#52 41 26 1544.5 1163 1288 1230 1170.5 1060 1047 364 731.5 729 NM_002358 LUA#53 33 32.5 564 409 570 611.5 616 671 583 275 547 464 NM_013402 LUA#54 34.5 31 1273.5 943.5 1181 1190 1216.5 1153.5 1108 456 976 945 NM_000875 LUA#55 42 30 1243.5 1137.5 1219.5 1507 1425 1383 1250 854.5 1158 1168 NM_001974 LUA#11 33 34 147 137 170 221.5 273 213.5 183 58 139 130 NM_000632 LUA#12 41 35 500.5 399 483 509 499.5 519.5 492 282 378 338.5 NM_006457 LUA#13 33.5 30 94 75 82.5 91 82 68 75 38 60 59 NM_000698 LUA#14 38.5 28 188 153 163.5 209 215.5 184 149 99 134 133 NM_032571 LUA#15 34.5 49.5 209 146 172 223 198 173.5 187 87 152 150 NM_006138 LUA#56 44 38.5 145.5 150 157 229 199 209 158 133 140 130 NM_015201 LUA#57 42 33 878 689 822 965 877.5 927 932 381 635 570 NM_006985 LUA#58 38 34 919 775 826 897 857 925 751 292.5 727 619 NM_004095 LUA#59 41 32 695 536.5 595 574 562 655 565.5 183.5 345 337.5 NM_005914 LUA#60 46 37 2195.5 1744 2157 2234 2262 2579 2082 1102 2212 2079 NM_007282 LUA#16 34 20 4387 3871 4222 4458 4248 4005 4536 3049.5 3935 3689 NM_003644 LUA#17 36 33 526 406 480.5 528 498 450 494 246.5 411.5 391.5 NM_001498 LUA#18 42 36 1913 1585 1809.5 2005 1957 1776.5 1849 805 1607 1538 NM_003172 LUA#19 39 33 3589 2978.5 3400 3500 3410.5 3151 3536 3020 3531 3474 NM_004723 LUA#20 60 48 832 591.5 736.5 873 807.5 813 798 329.5 716.5 652 NM_014366 LUA#61 38 28 1995 1551 1903.5 2057 1962 1912.5 1996 1294.5 1720 1635 NM_003581 LUA#62 38 39 360 341.5 317.5 455 640.5 540 412 151.5 429 402 NM_018115 LUA#63 38 31.5 3024 2378 2960 3112 2963 2980 2866 1873 2710 2595 NM_021974 LUA#64 36 35 2077.5 1654.5 2019 2122 2051 2001 1859.5 973.5 1770.5 1771 NM_024045 LUA#65 42 40 734 526.5 675 775 713 729 683 264 520 494 NM_004079 LUA#21 42 31 4089 3862.5 3968 3977 3945.5 3731 3760 2211 3375 3283.5 NM_000414 LUA#22 30.5 38.5 604 446 533 594 583 764 580 203 475 440.5 NM_001684 LUA#23 36 38 2409.5 1974 2345 2586 2361 2644 2639 1719.5 2063 2080 NM_003879 LUA#24 31 29.5 960 709.5 920 1061 1060.5 1079.5 920.5 446 891 871 NM_002166 LUA#25 41 29 1321.5 1026 1432 1466 1409 1475.5 1220 663.5 1490.5 1453 NM_005952 LUA#66 40 36 1423 1277.5 1395.5 1459.5 1482 1431 1332 675.5 1259 1185 NM_001034 LUA#67 40 36 607 491.5 520.5 777 713 635.5 580 255 614 609 NM_003132 LUA#68 36 42 789 626 706 671 617 563.5 583 198.5 518.5 524 NM_018164 LUA#69 41 34 205.5 149 182 235 274 250 198 100.5 189 142 NM_014573 LUA#70 41 39 292 225.5 240 328.5 314.5 272 244.5 114 257.5 232 NM_014333 LUA#26 28.5 27 1505 1147 1369.5 1467 1427 1484 1415 774.5 1217.5 1236 NM_006432 LUA#27 38.5 33 699 534 646 713.5 703 718 636 315 562 550 NM_000433 LUA#28 45 44 878 576 830.5 896 906 796 844 351 893 824 NM_000147 LUA#29 42 24 639 466 629 651 659 597.5 645 256 532.5 499 NM_000584 LUA#30 41.5 36 394 346 379 483.5 407 340.5 306.5 120 268.5 289 NM_006452 LUA#71 35 36 2704.5 2307.5 2678 2654.5 2673 2689 2707 1357.5 2109 1953 NM_005915 LUA#72 45.5 39 1061.5 874 1025 1120 1087 921 1013 478 1105 1020 NM_005980 LUA#73 40.5 44.5 159 108 139 145.5 144.5 144 145 92.5 138.5 130 NM_002539 LUA#74 47 43 2035.5 1756 2051.5 2189.5 2318 1930 1994 1204.5 2047.5 2038 NM_019058 LUA#75 48 37 2504 2473 2482 2914 3027.5 2942.5 2642 1576 2562.5 2616.5 NM_004152 LUA#31 44 42 1205 983 1218 1317 1344 1212 1299 547.5 1317.5 1129.5 NM_004602 LUA#32 38 30 182 293 205 255 222 159 170 770 223.5 139 NM_018890 LUA#33 51 44 2917 2521.5 2741.5 2699 3109 2785 3028.5 2194 2814 2125 NM_001101 LUA#34 47 41 3269.5 2707 3122.5 3280 3254.5 2939 3057 2117 3070 2979 NM_006019 LUA#35 40 32.5 732 617.5 657.5 710.5 678 550.5 633 242 493 479.5 NM_004134 LUA#76 53 49 1773 1613 1923 1777.5 1756.5 1565 1674 812.5 1734 1752 NM_005008 LUA#77 38 37 1466 1175 1420 1489 1546 1279 1331 613.5 1198 1154 NM_020117 LUA#78 37 32.5 3623 3228 3691 3649 3820 3251 3418 2516 3693.5 3553 NM_001469 LUA#79 35 30.5 609.5 490 632.5 745 811 727 615.5 295 646 600 NM_021203 LUA#80 43 48 854.5 657 825 824 830.5 702 752 289.5 812 729 NM_002624 LUA#36 54 45.5 483 414 462 482.5 490 414.5 426 178 314 300.5 NM_004759 LUA#37 45 40 210 160 207 214.5 192 162 157 97 190 175 NM_002664 LUA#38 42.5 44 758.5 572 687 715.5 717 676 717 272 683 690 NM_000211 LUA#39 43 47 2399 2085 2457.5 2480 2328 1741 2234 1125 2855 2765 NM_002468 LUA#40 36 41.5 434 421 408.5 461 466 408 403 238 396.5 335 NM_000884 LUA#81 48 53 1425.5 1158 1403 1476 1501.5 1293 1396 661 1224.5 1201.5 NM_003752 LUA#82 51 46 2178 1591 1908 2000 2057 1847 2035 1041.5 1743 1589 NM_018256 LUA#83 38 42 1960 1487 1947.5 1945.5 1933 1831 1798 1027 1858.5 1781 NM_001948 LUA#84 51 44 3639 3037 3513 3628 3641 3222 3639 1898.5 3089 3064 NM_005566 LUA#85 50 46.5 2849 2508 2754 2860 2845 2649 2739.5 1334 2560 2497 NM_021103 LUA#41 51 45 3369.5 2796 3116 3286.5 3175 2888 3034 2155 2887 2663.5 NM_002970 LUA#42 50 53 1390 1330 1252 1169.5 1144.5 922.5 1002 381 800.5 798 NM_003332 LUA#43 37 42 3442 3303 2960 2860 2644 2238 2494 1006 1976 2066.5 NM_004106 LUA#44 43 40 756 623 688 662 601 546 562 203 416.5 430.5 NM_002982 LUA#45 48 38.5 4465 4583 4733 4626 4576 4067.5 4536 2998 4098 3942.5 NM_005375 LUA#86 53.5 53 3445 2883 3140 3429 3216 3079 3213.5 1598.5 2714 2510 NM_000250 LUA#87 50 40.5 3990 3233.5 3862 3996 3850 3694.5 3993 2672 3456 3368 NM_004526 LUA#88 42 31 2129 1933 2176 2149 2161 1926 1970.5 1115 1947 1890.5 NM_004741 LUA#89 50.5 39 1970 1864 1808.5 1645 1661 1432.5 1528 561.5 1340 1146.5 NM_002467 LUA#90 67 56.5 3253 2824 3142 3156.5 3104 2666 2784 1819.5 2700.5 2541 ACTB LUA#91 54 51 3126 2638 3086 3191 3160 3024 3100 1853.5 3149 3002.5 TFRC LUA#92 76 79.5 1348 983.5 1283.5 1329.5 1267 1098 1256 467.5 946 967 GAPDH_5 LUA#93 59 46 2708 1911 2385.5 2693 2523 2374.5 2539.5 1475 2364 2243.5 GAPDH_M LUA#94 48 49.5 4772 3907 4477 5031.5 4540 4282 4848 3529 4163 4180 GAPDH_3 LUA#95 74.5 69 4277 3837 4461.5 4434 4414 4444 4482 3794.5 4211 4058 Table 5B. Microtiter plates FlexMap description ID dmso11 dmso12 dmso13 dmso14 dmso15 dmso16 dmso17 dmso18 dmso19 dmso20 dmso21 dmso22 NM_005736 LUA#1 863 780.5 645 792.5 662 690 686.5 690 744 752 821 824.5 NM_000070 LUA#2 602 551 497.5 605 489 519 524.5 532 532.5 541 574 575 NM_018217 LUA#3 1301 1291 1131 1309.5 1049 1136 1159 1144 1216.5 1295 1334 1278 NM_004782 LUA#4 1261.5 1219 1206 1280 936.5 1113 1077 1085 1223 1228 1291.5 1200 NM_014962 LUA#5 1351 1339 1064 1149.5 1037 1121 1101 1135 1245 1246.5 1325 1246.5 NM_004514 LUA#46 1269 1286.5 1143 1367 1083 1216 1144 1196 1271 1302 1276 1284.5 NM_006773 LUA#47 742.5 671 677.5 757 598 690.5 691.5 689 687 707 730 706 NM_014288 LUA#48 754 671 683 764 579 735.5 701 704 708 718 733 708 NM_017440 LUA#49 533 498 481.5 569 436 529 490 506 499 527 533 544.5 NM_007331 LUA#50 756 792 605 745 636 718 726 692 711 767 785 786 NM_173823 LUA#6 876 1030 673.5 802 672 763 735 738 861.5 954 913.5 959 NM_000962 LUA#7 293 363 281 328.5 281.5 275 278 278 291 340 348 342 NM_003825 LUA#8 350 335 293 267.5 254 222 245.5 265 313 315 347 310 NM_016061 LUA#9 737 740 530.5 653.5 623 649 597 618 659 648 707 681 NM_000153 LUA#10 44.5 46 46 44 39 41 44 42 43 50 53 51 NM_006948 LUA#51 51 55.5 56 65 56 62 55 60 55.5 64 57 60.5 NM_004631 LUA#52 792.5 864 593.5 702.5 575 698.5 641 698.5 709.5 756 744.5 779 NM_002358 LUA#53 614 582.5 560 676 542.5 606 503 526 553 560.5 578.5 574.5 NM_013402 LUA#54 974 1061 870 999 812 940.5 906.5 918 941 970.5 977 1031 NM_000875 LUA#55 1337 1263 1215 1372.5 1168 1101 1141.5 1096 1191 1173.5 1280 1223 NM_001974 LUA#11 194 214 175 216 163.5 117 114 119 164 178 222.5 205.5 NM_000632 LUA#12 360 389.5 361 404 333 383 372 307 361.5 376 396 397 NM_006457 LUA#13 71 62.5 56 64.5 55 56.5 50 60 67 65 67 64 NM_000698 LUA#14 132.5 136 123.5 166.5 146 130 114 129 115.5 135 142 145 NM_032571 LUA#15 132.5 173 141 190 108 160.5 135 142.5 164.5 170 178 157 NM_006138 LUA#56 128 133.5 142 134 140 138 137 117 146.5 150 154 153 NM_015201 LUA#57 688 692 583 736.5 650 684.5 588.5 557 637 652 691.5 698 NM_006985 LUA#58 630 701 543.5 707.5 550 672 684 635 653.5 678 720.5 692 NM_004095 LUA#59 334 407 294.5 363 352.5 440 347.5 319 372 340 398.5 391 NM_005914 LUA#60 1967.5 2255 1967 2196 1708 2021 2120 1877 2054 2334 2477 2222 NM_007282 LUA#16 4208 4000.5 3735 4128 3643 3554 3724 3707 4109 3898.5 4083 3866 NM_003644 LUA#17 461 445.5 422.5 467 331 409 394 418 430 437.5 462.5 465.5 NM_001498 LUA#18 1627.5 1631 1477 1773 1383 1618.5 1582 1614 1701 1727 1700 1716 NM_003172 LUA#19 3838 3647 3528 3823.5 3374 3493 3499.5 3566 3683 3672 3821 3575 NM_004723 LUA#20 848.5 770 717 823.5 607 677 702.5 717 709 705 759 789.5 NM_014366 LUA#61 2015 1794.5 1782 2122.5 1726.5 1787 1758 1753 1799 1782 1903 1815 NM_003581 LUA#62 561 312 364 462 507 198.5 275 245 268 472.5 540 562.5 NM_018115 LUA#63 2942 2980 2750 3020 2659.5 2912 2714 2598 2775 2741 2898 2815 NM_021974 LUA#64 1949 1868 1777 2001 1535.5 1806 1739.5 1752 1837 1744 1888 1869.5 NM_024045 LUA#65 520 585 448 599 494 545.5 509 475 489 513.5 539.5 530 NM_004079 LUA#21 3630 3578.5 3061 3459 3268.5 3368 3393.5 3216 3473.5 3414 3469.5 3382 NM_000414 LUA#22 554 514 473 566 440 552 539 518 523 534 546.5 539 NM_001684 LUA#23 2436 2350 2186 2429 2206 2209 2068 2000 2280 2214.5 2479 2192.5 NM_003879 LUA#24 897 985 843.5 1017 839 918.5 909 959 942.5 961 1003 983 NM_002166 LUA#25 1616.5 1692 1460 1463 1050.5 1282 1493 1420 1532 1618 1690 1601 NM_005952 LUA#66 1343.5 1432.5 1069 1249 1130.5 1206.5 1195.5 1107.5 1166.5 1214 1263.5 1259 NM_001034 LUA#67 537 642 588 647 495.5 491 523.5 545 572 667 680 675.5 NM_003132 LUA#68 457 536 413 517 403 507 516 462 529 548 538 522 NM_018164 LUA#69 195.5 184 178 231 184 122 129 142.5 186 209.5 214 203 NM_014573 LUA#70 230 271 230 293 193.5 212 214 230 212 256 280 259 NM_014333 LUA#26 1335 1361 1221.5 1387 1155 1214.5 1230 1281 1305 1393 1462 1429 NM_006432 LUA#27 585 632 533 689 499 575 534 545 594 662 702.5 671.5 NM_000433 LUA#28 920 893 911 1009 655 928.5 927 928.5 950.5 969 1001 979 NM_000147 LUA#29 500 521 468 541 462 505 484.5 459.5 511.5 506 555 539 NM_000584 LUA#30 256 366 256 269 251 183 169.5 207 243.5 316 301.5 315 NM_006452 LUA#71 2099 2084 1892 2120 2006 2266 2093 1950 2197 2076.5 2209 2167 NM_005915 LUA#72 1226 1099 1053 1205.5 860 943 1063 1093.5 1138 1053 1123.5 1079 NM_005980 LUA#73 142 132 131 147 131 150.5 147.5 140 142.5 157 140 144 NM_002539 LUA#74 2425 2316 2087 2311 1877 1927.5 2018 1928 2145 2151 2222 2192 NM_019058 LUA#75 3031 2880 2490.5 2668 2516.5 2095 2271 2515 2626 2535 2676 2717 NM_004152 LUA#31 1242.5 1194 1192 1395.5 1060 1213 1238 1194.5 1259 1273 1272.5 1273 NM_004602 LUA#32 160 171 118 146 139 185.5 224 144 136 148 144 175 NM_018890 LUA#33 3178 2820 2759 3652 2563.5 2013 2134 1994.5 2953 3108 3381 3102.5 NM_001101 LUA#34 3390 3286 3055 3351 3058 2997 3223 3069 3164 3182.5 3260 3244.5 NM_006019 LUA#35 429 517 421 502 432 439 465 472 469 520 559 517.5 NM_004134 LUA#76 1839 1854.5 1599 1770 1402 1666 1823 1718 1844 1891.5 1836 1747.5 NM_005008 LUA#77 1088 1303 1122.5 1276.5 1020 1110 1139.5 1151.5 1213 1281 1304 1340 NM_020117 LUA#78 4107 3817 3879 4057 3458.5 3506 3738 3565.5 3943 3851.5 4059.5 3820.5 NM_001469 LUA#79 710.5 619 678 842 686 630 612 622.5 670.5 688 825 835 NM_021203 LUA#80 780 794 768 808.5 590 757 807 745.5 808 803 818 784.5 NM_002624 LUA#36 336 353 250 305 275 297 283 299 334.5 335 381 331 NM_004759 LUA#37 186 205.5 183 212 157 194 186 173 184 194 197 206 NM_002664 LUA#38 797 730.5 691 732 548 671 688 703 750 757 769 762 NM_000211 LUA#39 3211 2924 2886 2921 1857 2278 2657 2797 3053 2908 3039.5 2797.5 NM_002468 LUA#40 429 379.5 347 429 297 306.5 343 339 349 375 391 371.5 NM_000884 LUA#81 1428 1318 1197 1324 1090 1199 1217 1234 1315 1314.5 1350 1293 NM_003752 LUA#82 1846 1808.5 1603 1888 1612 1740.5 1728.5 1633 1761 1702 1825 1762 NM_018256 LUA#83 2062 1861 1845.5 2074.5 1645 1792 1851 1876 1910 1907 1960 1898.5 NM_001948 LUA#84 3418 3494 3142.5 3336 2664 2977 3066 3045 3170 3332 3464 3272.5 NM_005566 LUA#85 2977 2714 2584 2752 2214 2342 2574 2571 2654.5 2695 2715 2669.5 NM_021103 LUA#41 3189.5 3018 2718 3105 2604.5 2742 2818 2882 2966 2956 3130 2913.5 NM_002970 LUA#42 879 899 596 638 621 698 698 707 813 778 840 748 NM_003332 LUA#43 2000 2210 1489 1631.5 1664.5 1829 1865 1854 2095.5 2120.5 2375 2163 NM_004106 LUA#44 416.5 450.5 371 410 366 407 398 375 392 398 463 418 NM_002982 LUA#45 4022 4124 3811.5 4093 3650 3735 3781.5 3873 4035 4073 4216 3981.5 NM_005375 LUA#86 3004.5 2906 2558 2880 2360 2568 2646 2638.5 2846 2892 3039.5 2842 NM_000250 LUA#87 3741.5 3571 3474 3656 3421.5 3371 3432 3378 3509 3505 3695 3495 NM_004526 LUA#88 2058 2055 1808 1911 1680 1726.5 1825.5 1736 1909.5 1896.5 1978 1990 NM_004741 LUA#89 1108 1321.5 947.5 1238 1024 1083 1073 1051.5 1149 1280 1378.5 1306 NM_002467 LUA#90 2459.5 2556 2463.5 2716 2442.5 2612 2700 2639 2735 2770 2847 2864.5 ACTB LUA#91 3366 3226 2978 3292 2667 3186 3158 3128 3408.5 3183 3323 3238.5 TFRC LUA#92 948 1112 883 1059 758 1009 944.5 929 1063.5 1069 1197 1157 GAPDH_5 LUA#93 2063 2310 2363 2598 2157 2324 2337 2442 2468 2425.5 2655 2417 GAPDH_M LUA#94 4206 4269 4371 4733.5 4179.5 4071 4252.5 4207 4413 4315 4737 4324.5 GAPDH_3 LUA#95 4477 4343.5 4445 4632 3923 4014 4259.5 4169 4620.5 4371 4726 4365 Table 5C. Microtiter plates FlexMap description ID dmso23 dmso24 dmso25 dmso26 dmso27 dmso28 dmso29 dmso30 dmso31 dmso32 dmso33 dmso34 NM_005736 LUA#1 821.5 761.5 188 697 774.5 787.5 819 983.5 981.5 798 306.5 708 NM_000070 LUA#2 594.5 430.5 145.5 562 569.5 544.5 596.5 671 648 486 165 548.5 NM_018217 LUA#3 1272 1058 376 1157 1280 1212 1311 1475 1368 1128 433 1123 NM_004782 LUA#4 1254 1072 442 1106 1257 1209.5 1279 1435 1295 1042 496.5 1128.5 NM_014962 LUA#5 1284.5 950 381 1032 1210 1259.5 1287 1466 1347 1046 405 1094 NM_004514 LUA#46 1275 1119 432 1216 1259 1206 1358 1503.5 1391.5 1179 512 1155.5 NM_006773 LUA#47 691 616 242 666 731 726 757 756 731 663 283 684 NM_014288 LUA#48 701 566 240 703 741 758 751 738 705 616 285 687 NM_017440 LUA#49 568.5 487 184.5 503.5 534 536.5 553 615 619 504 214 489.5 NM_007331 LUA#50 842 569.5 172 659 721 712.5 770.5 918 866 625 207 673 NM_173823 LUA#6 1025 607 154 705 814 832.5 938 1231 1222 732 173.5 845 NM_000962 LUA#7 352 211 64 253 284 279 369 400.5 441.5 264 78 293 NM_003825 LUA#8 381 251.5 111 235 283 306 350 401 379 249 117 325.5 NM_016061 LUA#9 745 481 166 546 662 645 728 788 830 574.5 181 539 NM_000153 LUA#10 55 45 32 43 43.5 43 54.5 62 65 51 37 44.5 NM_006948 LUA#51 81 46.5 45 62.5 59 53 70 75 73 70.5 34 62.5 NM_004631 LUA#52 856 460 151 651.5 676 654 697 822.5 826.5 502.5 148 646 NM_002358 LUA#53 656 440 130 575 518 562 675 706 732 536 162 547 NM_013402 LUA#54 1023.5 761 223 871 927 926.5 975 1112 1116 832 250 883 NM_000875 LUA#55 1365 1238 416 1061 1243 1287 1314 1444 1351 1231 477.5 1182.5 NM_001974 LUA#11 210 101.5 49.5 148 138 150 217 378.5 312.5 119 51.5 131.5 NM_000632 LUA#12 422 322.5 70 324.5 355 343.5 371 420 466 365 101.5 346 NM_006457 LUA#13 82 45 39 55 64 67 67.5 89 100 51 37 62 NM_000698 LUA#14 196 141 53 133 119 131 158 214 204 156 49.5 135 NM_032571 LUA#15 171 126 43 146 184 147 184 200 220 135.5 54 161 NM_006138 LUA#56 187.5 154.5 53.5 125.5 140 118.5 156.5 179 181 152 66 140.5 NM_015201 LUA#57 785 654 157 602 652 676.5 745 864 989.5 753 187 597 NM_006985 LUA#58 748 480 115.5 632.5 634 596 693 721 753.5 549.5 136 577 NM_004095 LUA#59 449 277 74 298 339 355.5 377.5 423 534 335 97.5 282 NM_005914 LUA#60 2065 1570 585.5 1976 2363 2060 2352 2412.5 2143 1828 640 1900 NM_007282 LUA#16 3898 3815 2119 3519 4076.5 4109 4055 4442 4132 3867 2338 3559 NM_003644 LUA#17 463 361 151 404.5 468 438.5 476 510 481 365 173.5 435 NM_001498 LUA#18 1764 1346.5 396 1556.5 1713 1626 1775 1908 1881 1507 461 1600.5 NM_003172 LUA#19 3530.5 3727 2201 3535 3813 3727 3844 3804 3566 3747 2476 3638.5 NM_004723 LUA#20 733.5 581 164.5 714 744 778 808 839.5 848 691.5 205 742 NM_014366 LUA#61 1790 1932 755 1697 1841 1830 1930 1971 1885.5 2015 854 1815.5 NM_003581 LUA#62 508 264 59 336.5 263 336 362 671 472 392 100 295 NM_018115 LUA#63 2891 2754 1010 2590 2914.5 2749 3009 3130 3163 2849 1137 2663.5 NM_021974 LUA#64 1800 1540 533 1673 1868 1801 1844 1955 1839.5 1621 613 1682.5 NM_024045 LUA#65 580.5 456 127 458 493 477 564.5 602 684.5 541 149 490 NM_004079 LUA#21 3373 2792 1173 3108.5 3361 3403 3373 3610.5 3401 2919 1179 3060 NM_000414 LUA#22 573 384 101 508 570.5 556 556 574.5 625 456 113 509.5 NM_001684 LUA#23 2316 2260 966 2120.5 2395 2329.5 2457 2669.5 2647.5 2428 1083 2158 NM_003879 LUA#24 919 761 233 892.5 963 916.5 985 1092 1063 893 283.5 903 NM_002166 LUA#25 1348 993 408 1513 1746 1565 1930 1844 1355 1134 467 1441.5 NM_005952 LUA#66 1283 1016.5 336 1102 1159 1200 1283 1387 1325 1101 353 1138 NM_001034 LUA#67 689.5 450 112 505 540 557 672 811.5 809 423 132 611 NM_003132 LUA#68 535 319 94 458.5 473 475 503 592 559 372 105 444 NM_018164 LUA#69 226 130 59 149 147 157 221.5 281.5 252 165 63 160.5 NM_014573 LUA#70 277 201.5 61.5 219 207 238 288.5 333 436 224 73 237 NM_014333 LUA#26 1363.5 1109 448 1216 1325 1285.5 1464.5 1547 1503.5 1192 521 1233 NM_006432 LUA#27 716.5 478 170.5 548.5 613 585 701 828 774.5 545.5 207.5 565.5 NM_000433 LUA#28 900 625 185 906 976.5 938 971.5 1056 926 710 226.5 875 NM_000147 LUA#29 565 447 136 456 509 496 566 633.5 674 499 160 509 NM_000584 LUA#30 332 167.5 51.5 194 216 250 440.5 577.5 679 232 64 266.5 NM_006452 LUA#71 2382 1862 572 1894.5 2078 2134 2062 2363 2464.5 1959 642 1927 NM_005915 LUA#72 1040.5 812 258 1015 1145 1113 1142 1191 1078 905.5 298 1096.5 NM_005980 LUA#73 162 113.5 46 145.5 150 140 143.5 142 143.5 133.5 65 134 NM_002539 LUA#74 2219.5 1712 716.5 1940 2176 2201 2248 2379 2236 1902 756 1957.5 NM_019058 LUA#75 2565.5 2239 883 2338.5 2445 2617 2767.5 2997 2585 2218.5 937.5 2571 NM_004152 LUA#31 1238.5 885 254.5 1116 1248 1222 1313 1419 1325.5 1031 326 1154.5 NM_004602 LUA#32 207.5 581 86 127.5 144 138 172 214 245 385 210 165.5 NM_018890 LUA#33 3131.5 2053.5 683 2273 2061 2264 3223 3293 2669 2625 1280 1831 NM_001101 LUA#34 3138 2898 1256 2946 3193.5 3261 3363 3638 3259 3136 1401 3093.5 NM_006019 LUA#35 552 373 118 421 443 456 541 679 653 420.5 131 419 NM_004134 LUA#76 1621 1208 429 1734 1827 1817 1814.5 1856 1730 1437 510.5 1704.5 NM_005008 LUA#77 1325 876 284 1091 1131.5 1088 1258 1464 1405.5 945 321 1145 NM_020117 LUA#78 3812 3366 1892.5 3512 3795 3850 3953 4053.5 3597.5 3343.5 2066 3664 NM_001469 LUA#79 816 489 131.5 644 627 637 698 1000 783 548 176 613.5 NM_021203 LUA#80 802 445 136 682 771.5 789.5 811 929 728 523 163.5 740.5 NM_002624 LUA#36 396 259.5 81 285 310 308.5 361 445 448 296 101 332.5 NM_004759 LUA#37 212 146 56 191.5 195 200 218 230 229 174 73 180 NM_002664 LUA#38 713 463 147 679 769.5 768 798 850 820 551 158 712 NM_000211 LUA#39 2300 1665 817 2789 3080.5 3084 3060 3115 2385.5 1682 880 2773 NM_002468 LUA#40 427 289 84 328.5 354 368 391.5 437 500 341 105 374.5 NM_000884 LUA#81 1285 948 318 1168 1347 1357 1357.5 1526.5 1366 1084 349 1198 NM_003752 LUA#82 1763 1642 538.5 1556 1773 1728.5 1820 2059 2038 1723 603.5 1651 NM_018256 LUA#83 1856 1542 566.5 1765 1956.5 2005 1978 2084.5 1880 1678 628 1815.5 NM_001948 LUA#84 3267 2654 1083 2928 3321 3331 3460 3698 3453.5 2621.5 1165.5 3034 NM_005566 LUA#85 2590 1917.5 682 2426 2711 2775 2726 2956 2650.5 2079 728 2471 NM_021103 LUA#41 2956 2604.5 1342 2649 2997 3023.5 3125.5 3151 2850 2606 1390 2874 NM_002970 LUA#42 879 470.5 177 617 645 720.5 854 935 819 484 175 626 NM_003332 LUA#43 2270 1228 527 1706 2044 2248.5 2272.5 2640 2476.5 1319.5 496.5 1835.5 NM_004106 LUA#44 486 268 100 347 378.5 379 441.5 519 499.5 309 101.5 338 NM_002982 LUA#45 4008 3520 1385.5 3498 3857.5 3867 3911.5 4376.5 4090 3402 1612 3652 NM_005375 LUA#86 2929 2138 780 2591 3000 3007 2930 3132 3068 2187 846.5 2508 NM_000250 LUA#87 3651 3489 1765 3299 3612 3693 3847 4025 3686.5 3647 1803 3408 NM_004526 LUA#88 1880 1497 585 1628 1882 1926 2035.5 2119 2056 1644 650 1709 NM_004741 LUA#89 1381 700 242 992.5 1014 1103 1378 1416 1429 794 253 998.5 NM_002467 LUA#90 2628 2183.5 955 2420 2720 2741 2784 2979 2694 2237.5 1032 2422 ACTB LUA#91 3135 2568 1118.5 3053.5 3423.5 3204 3422 3556 3070 2801.5 1285 3053 TFRC LUA#92 1174 664 207 912 1113 1032 1189 1370 1388 813 235 1034 GAPDH_5 LUA#93 2447 2014.5 859 2239.5 2438 2261 2400 2572 2390.5 2139 1023 2433 GAPDH_M LUA#94 4314 4528 2358.5 4048 4414 4150 4464 4643 4474 4483.5 2639.5 4111 GAPDH_3 LUA#95 4468 4283 3479 3998 4500 4518 4621 4645.5 4414 4249 3411 4026 Table 5D. Microtiter plates FlexMap description ID dmso35 dmso36 dmso37 dmso38 dmso39 dmso40 dmso41 dmso42 dmso43 dmso44 dmso45 dmso46 NM_005736 LUA#1 800 833 740.5 838.5 652 751.5 746 714.5 87 136 806 835 NM_000070 LUA#2 605.5 588.5 578.5 652.5 538 377.5 350 518 67 62.5 534.5 556 NM_018217 LUA#3 1308.5 1279.5 1242 1243.5 1030 901 915 1109.5 89 167 1158.5 1114 NM_004782 LUA#4 1238 1228 1232 1133 974 882.5 885.5 1085 96 187.5 1077.5 1018 NM_014962 LUA#5 1158 1208 1175 1187 1015 823 819 1036 87 161 1104 1084 NM_004514 LUA#46 1237 1258 1186 1216 1067.5 909 946 1072 88 178 1161 1144 NM_006773 LUA#47 769 741 699.5 701.5 619 544 528 685.5 88 128 653 647 NM_014288 LUA#48 733 721.5 667 692 609.5 478 510 676 132 140.5 643.5 702 NM_017440 LUA#49 582 547 529 527 462 423 387 490 73 97.5 501 562 NM_007331 LUA#50 744 743 749.5 756 670.5 465 464 657 66 92 679 711.5 NM_173823 LUA#6 761 855 839 836 801 520.5 491.5 695 47 64 894 880 NM_000962 LUA#7 309 343.5 293 332 297 178 186 245 44 30 295 316.5 NM_003825 LUA#8 286 240 257 299 278.5 182 215.5 256 62 72 306 331 NM_016061 LUA#9 586 658.5 613 659 536 369 398.5 507 66 83.5 557.5 606 NM_000153 LUA#10 47.5 56 50 57 56 46 40.5 41 29 28 54 64 NM_006948 LUA#51 62 61.5 69.5 67 67 56 51 51 29 15 57.5 71 NM_004631 LUA#52 643.5 646 686.5 663 591 328 336 545 80 92.5 615 650 NM_002358 LUA#53 573.5 540 564 592 580 419 385 538 36.5 49 559 606.5 NM_013402 LUA#54 966 978 921 940.5 856.5 563.5 599 823 46 95 875 880 NM_000875 LUA#55 1285.5 1133 1138 1263 1075 1092 1048 1124 106 188.5 1221 1110 NM_001974 LUA#11 138 141 155.5 212 134 83 93 119 36 35 137 207 NM_000632 LUA#12 387 363 342 400 356 348 296 342 47.5 53 353 359.5 NM_006457 LUA#13 62 71.5 61 81 78.5 55 49 48.5 28 23 75 63 NM_000698 LUA#14 146 141 122 167 160.5 135 125.5 121.5 40 33.5 148 200 NM_032571 LUA#15 164 176.5 167.5 169 138 110 109 129.5 28 33 160 172 NM_006138 LUA#56 166.5 146 121 158.5 152 141.5 125 125 39 46 135 151 NM_015201 LUA#57 686.5 706 631.5 729 656 569 485 618 42 74 666 624 NM_006985 LUA#58 638 615 623 582 538 345 345 555 37 54 584 588 NM_004095 LUA#59 304 350 338.5 374 354 235 211 281 38 46 316 357 NM_005914 LUA#60 2448.5 2103 2338 1967.5 1571 1343 1339 2015 118 230 1893 1677 NM_007282 LUA#16 3893.5 3874.5 3637.5 3391 3071 3437 3494 3535 359 966 3373 3307 NM_003644 LUA#17 446 449.5 439 424 365.5 311 308.5 406 53 79 411 413 NM_001498 LUA#18 1703 1725 1714 1637 1450 1059 1094 1550 69 141 1618 1541 NM_003172 LUA#19 3764 3726.5 3602 3543 2943 3368 3715.5 3362 481 1067 3343 3330 NM_004723 LUA#20 813.5 783 707 724 636 453 457 671.5 41 77 685 708 NM_014366 LUA#61 1911 1754 1710 1737.5 1493 1770 1731 1733 126 331.5 1643 1713 NM_003581 LUA#62 257.5 265 351 494.5 436 221.5 299 340 41 42 405 615.5 NM_018115 LUA#63 2928.5 2916 2747 2814 2454.5 2212 2288.5 2701 162.5 384 2539.5 2803 NM_021974 LUA#64 1901 1937 1813 1847 1596 1284.5 1344 1669 113 212.5 1647.5 1703 NM_024045 LUA#65 479 524 444 568 465 367 340.5 409.5 40.5 56 475 450 NM_004079 LUA#21 3238 3361 3198 3133 2881 2391 2398 2889 181 455 3041.5 2926 NM_000414 LUA#22 568 553 535 523 514 336 297 495 40.5 46 491.5 489 NM_001684 LUA#23 2275 2323.5 2187 2171 1887.5 1959.5 1968 2061 177.5 450.5 2037.5 2024 NM_003879 LUA#24 986.5 968.5 943.5 939 784 585 663 892 49 93 827 895.5 NM_002166 LUA#25 1657 1602 1549 1381 965.5 726 980.5 1589 75 166 1280 1227.5 NM_005952 LUA#66 1260 1233.5 1097 1147 991 801.5 831 1068 58 116.5 1122.5 1144 NM_001034 LUA#67 626 529 574 618 505 295 404 608.5 45 58 619 704 NM_003132 LUA#68 482 469 474.5 473 389 253.5 259.5 423 46.5 46 408 387 NM_018164 LUA#69 170 161 167 261 164 111.5 136 151 45 39 201 256 NM_014573 LUA#70 267.5 271 267 275.5 244 160 170 247 32 43.5 243 207 NM_014333 LUA#26 1364.5 1335.5 1312 1377.5 1155 983 962.5 1199.5 104 191 1229.5 1285 NM_006432 LUA#27 642 639 657 680.5 551 408 416 547 59 84 598 584.5 NM_000433 LUA#28 1066 942 920 924 750 515 567.5 860 47 80 841 797 NM_000147 LUA#29 529 555.5 529 517 462.5 387 380 487.5 45 65 533 539 NM_000584 LUA#30 242 278 258 423 221 124 161 199.5 40 40 257.5 294 NM_006452 LUA#71 2013 2089 2061 1989 1848 1611.5 1467.5 1781 101 221.5 1882 1980.5 NM_005915 LUA#72 1188 1179 1051.5 1098 845 584.5 691 948 59 101 1030.5 1063 NM_005980 LUA#73 153 160 142 150.5 126.5 112 104 137 38 32 140.5 128 NM_002539 LUA#74 2191 2195 2121 2170 1808.5 1433 1564.5 1898 118 287.5 2001.5 1947 NM_019058 LUA#75 2782 2271.5 2318 2538.5 2171 1860 2008 2257 134 341 2417 2269 NM_004152 LUA#31 1261 1241 1153 1334 991 696 753 1089 54 100 1113 1186 NM_004602 LUA#32 265.5 213 162 220 199.5 625 660 131 93 90 275 257.5 NM_018890 LUA#33 2075 2485 2508.5 3390.5 1910 2162 2101 2009.5 165.5 458 2526 2784.5 NM_001101 LUA#34 3429.5 3266.5 3048 3076 2572 2528 2638 3090 204 538.5 2953 2968 NM_006019 LUA#35 466 541 465 530.5 460 286 331 389.5 40.5 51 471 498 NM_004134 LUA#76 1792.5 1804.5 1765 1703.5 1324 1003 1120 1633.5 80 164 1503.5 1611.5 NM_005008 LUA#77 1191 1314 1203 1286 965.5 702 700 1069 68 121 1117 1163 NM_020117 LUA#78 3834 3886 3716.5 3752.5 3058 2885 3210 3558 317 821.5 3341 3770 NM_001469 LUA#79 681 598.5 718.5 792 616 403 431 600 49 70 579.5 749 NM_021203 LUA#80 807 744 750 772 661 371 410 686 49 69 730.5 642.5 NM_002624 LUA#36 278 328.5 338 388 292 234 213.5 274.5 34 48.5 323.5 411 NM_004759 LUA#37 193 202 188 206 158 134 138 184.5 38 42 180 185 NM_002664 LUA#38 714 737 750 734 590 385.5 418 645.5 40 64.5 656 700.5 NM_000211 LUA#39 3006 2869 2683 2721 1875 1271 1745 2569 132 322.5 2468.5 2468 NM_002468 LUA#40 379 415 338.5 380 305 294 281.5 294 45 51.5 378 353.5 NM_000884 LUA#81 1287 1282 1226 1286.5 1040 779.5 865 1131.5 70 124 1225 1136 NM_003752 LUA#82 1821.5 1763 1615.5 1734 1487 1332.5 1338 1538 91.5 208.5 1630 1496 NM_018256 LUA#83 2020.5 1982 1850 1812.5 1542.5 1282 1341.5 1768 89 218.5 1730.5 1731 NM_001948 LUA#84 3271 3345 3206 3253 2653 2216.5 2299 2996 214 499 3014.5 2966 NM_005566 LUA#85 2684 2614.5 2520 2485 2077 1560 1596 2313 109 268 2317 2122.5 NM_021103 LUA#41 2997 2869 2675 2722 2340 2323.5 2451 2529 321 666 2658 2720.5 NM_002970 LUA#42 648 665.5 668 715 587.5 354 371 529 72 91 654.5 656 NM_003332 LUA#43 1942.5 2132 2127 2213 1879 953.5 987 1653.5 218.5 335 1969 1813 NM_004106 LUA#44 375 377.5 366 397.5 359 217.5 210 330 47 55 348 355 NM_002982 LUA#45 3896.5 3808 3711 3649 3206 3020 3081 3635 273 694 3579 3162 NM_005375 LUA#86 2763 2813 2692 2661.5 2436 1824 1784.5 2537 157 354 2526.5 2672 NM_000250 LUA#87 3517 3557 3405 3467 3013 3199.5 3142 3251 299 711 3253 3188.5 NM_004526 LUA#88 1885 1915 1804.5 1852 1579.5 1229 1326.5 1636 115 248 1701 1706.5 NM_004741 LUA#89 1002 1136 1118 1351 929.5 501 537.5 825 90 128 977.5 1228.5 NM_002467 LUA#90 2713 2738 2634 2516 2218 1932 1877 2262 270 462 2574 2413 ACTB LUA#91 3240 3312 3154 3122.5 2542.5 2334 2382.5 2809.5 185 452 2830 2846.5 TFRC LUA#92 1052 1166 1040 1153 979.5 598 566.5 952 71 108.5 1087 990 GAPDH_5 LUA#93 2458 2471 2312 2286 1881 1785.5 1872 2132 141.5 332 2197 1991.5 GAPDH_M LUA#94 4477.5 4376 3992.5 4130 3535.5 4220 4298 3887.5 405 961.5 3835 3521 GAPDH_3 LUA#95 4410 4411 4111.5 4179 3477.5 4067 4018.5 3937 1107 2164 3853 3345 Table 5E. Microtiter plates FlexMap description ID dmso47 tretinoin1 tretinoin2 tretinoin3 tretinoin4 tretinoin5 tretinoin6 tretinoin7 tretinoin8 tretinoin9 NM_005736 LUA#1 712.5 1007 600 745 120 784.5 969 868 403 1056 NM_000070 LUA#2 542 645 609.5 617.5 257 804.5 748 679 244 752 NM_018217 LUA#3 972 1449 1280.5 1420 201 1539.5 1583 1510 682.5 1494.5 NM_004782 LUA#4 880.5 1159.5 1019.5 1093 191.5 1254 1263 1211.5 610.5 1219.5 NM_014962 LUA#5 1037 1464 1254 1316.5 176 1544 1556 1381 600 1344 NM_004514 LUA#46 941 1137 1091 1095 124 1305 1280 1218 659 1230.5 NM_006773 LUA#47 518 891 958 980.5 376 1067 994 1039 537 1062.5 NM_014288 LUA#48 524 640 712 763.5 370 801 816 737 395.5 809 NM_017440 LUA#49 461 544 516 515 226.5 586 612.5 615 298 622 NM_007331 LUA#50 638.5 912 911 865 183 1163.5 1068 987 369 958 NM_173823 LUA#6 960 1186.5 1029 1067 66 1345 1453 1179 381.5 1145.5 NM_000962 LUA#7 353 753 749 829.5 56 863 827.5 775 259 910.5 NM_003825 LUA#8 399 472 311 338 90 452 463 392 149 374 NM_016061 LUA#9 615 1280 1287 1337 110 1411 1519 1429 611 1267 NM_000153 LUA#10 119 141 148 144 44 160 184 146 57 152 NM_006948 LUA#51 75 75.5 64.5 65.5 37.5 75 66 94 47 67.5 NM_004631 LUA#52 651 893.5 845 865 133 1055 1218 998.5 283 808 NM_002358 LUA#53 498 418.5 426 405 34 477 491 522 210.5 523 NM_013402 LUA#54 789 1188.5 1164 1216 51 1393.5 1428 1345 506 1246 NM_000875 LUA#55 958 1248 1018.5 1094.5 75 1151.5 1201 1151.5 672 1198 NM_001974 LUA#11 198 826 132 221 30 240 313 382 72 590 NM_000632 LUA#12 363 485.5 406 446.5 45.5 519 580 537 172 496.5 NM_006457 LUA#13 135 83 79 67 36 91 109.5 88.5 38 81 NM_000698 LUA#14 220 252 202 222 47 259 284.5 292 92 236 NM_032571 LUA#15 191 193 192 197 53 236 253 217 71 239 NM_006138 LUA#56 210 557 420 445 45 467.5 500 464 203 494 NM_015201 LUA#57 705.5 1456 1263 1605 73 1699.5 1741 1620 797 1647.5 NM_006985 LUA#58 486.5 1364 1663 1539 48 1704.5 1609 1657 540 1438.5 NM_004095 LUA#59 376 714 733 799.5 43 891 900 902 277 764 NM_005914 LUA#60 1384 1942 1765.5 1972 209 2367 2086.5 2213 1002 1994 NM_007282 LUA#16 2507 3727.5 3308 3659.5 148 4025.5 3945 3663.5 2556 3643 NM_003644 LUA#17 374.5 374 336 376 136.5 402.5 436 387.5 203 400 NM_001498 LUA#18 1440.5 1427 1476.5 1522.5 89 1721 1766 1670 620 1578 NM_003172 LUA#19 2385.5 3240 3377 3457 142 3452 3345.5 3194.5 2743 3711 NM_004723 LUA#20 588 977 863 1030.5 44 1074 1047 982 435 1148 NM_014366 LUA#61 1280.5 1716 1736 1892 51.5 1915 1973 1899 1422.5 1937.5 NM_003581 LUA#62 345 742 360 455 48 551 988 918.5 186 626.5 NM_018115 LUA#63 2140 3715 3778.5 3863 104 3963 3999.5 3870.5 2808.5 3954 NM_021974 LUA#64 1382 2119 2344.5 2289 107.5 2544 2617.5 2309 1258 2411.5 NM_024045 LUA#65 484 771 761 793 47 917 904 960.5 346 825 NM_004079 LUA#21 2374.5 3579.5 3604 3848 137.5 4150 4022 3854 1810 3690.5 NM_000414 LUA#22 504.5 669 806.5 897 37 930.5 889.5 848 319 954 NM_001684 LUA#23 1613 3259 2761.5 3205 115 3451 3522 3269 2585 3440 NM_003879 LUA#24 707 1579.5 1854 1864 56 2010 2086 1929 996 1963 NM_002166 LUA#25 838 2678.5 2699 3180 82 2976 2983 2559 1905 3511.5 NM_005952 LUA#66 943 957 924 940 58 976 1108 1027.5 375.5 941 NM_001034 LUA#67 554 891 421 558 64.5 662 644 688 186.5 824 NM_003132 LUA#68 411.5 374 402.5 388 53.5 506 493 404 97.5 371 NM_018164 LUA#69 207 258 161 205 45 239 301 343 88.5 244 NM_014573 LUA#70 283 446.5 172 196 52 244 306 260 86 369 NM_014333 LUA#26 1010.5 1288 1167 1274.5 249 1456.5 1539 1513 672.5 1394.5 NM_006432 LUA#27 528 663 465 539 116.5 748.5 749 805 261 687 NM_000433 LUA#28 594 353 431.5 443.5 37 540 453.5 429 158 476.5 NM_000147 LUA#29 472 271 214 261 49 313 299 265 94 297.5 NM_000584 LUA#30 380 1027 270 453 50 411 600 505.5 116 723 NM_006452 LUA#71 1689 1715 1680 1742.5 83.5 2089 2031 1986 764 1717 NM_005915 LUA#72 768 481 443 497.5 50 517 560 541 173 543 NM_005980 LUA#73 147 106 90 96 46 92.5 99 112 47 90 NM_002539 LUA#74 1486 794.5 825 806.5 62.5 906 907 906 341 879 NM_019058 LUA#75 1861 2700.5 2316 2808 62.5 2607 2827 2598 1106 2635 NM_004152 LUA#31 844 613.5 664 635.5 50 804.5 811 920 225 661 NM_004602 LUA#32 439 967.5 114 307 49 178.5 275.5 231 315 364 NM_018890 LUA#33 1456 3289.5 2445.5 3142 144 3827.5 3781 4048.5 1671 3276.5 NM_001101 LUA#34 2148 2044 2141 2169 72 2194 2152 2208 1143.5 2249.5 NM_006019 LUA#35 475.5 431 378 402 61 452 533 447 108 403 NM_004134 LUA#76 1078 1012 1176 1010.5 67 1224.5 1261.5 1071.5 361 1060 NM_005008 LUA#77 895 1088 865 951 60 1096.5 1252.5 1117.5 281 1095 NM_020117 LUA#78 2483 2041 2196.5 2274 75.5 2432 2308 2248 1261 2246 NM_001469 LUA#79 496.5 850 405 467.5 47 592.5 796 833 164 637 NM_021203 LUA#80 559 396.5 401 428 50 487 504 437.5 92 406 NM_002624 LUA#36 354.5 378 268 348 52 451.5 542.5 417 124 342.5 NM_004759 LUA#37 183.5 130 145 140 49 150.5 165 169 45.5 133 NM_002664 LUA#38 573 797 806 872 56.5 987 938.5 870 293 950.5 NM_000211 LUA#39 1417 1438.5 1493 1537 64 1639 1647 1273.5 446 1558 NM_002468 LUA#40 370 463 273 333 55 355 441 352.5 117.5 387 NM_000884 LUA#81 967 907 802.5 882 79 1048.5 1027.5 931 331.5 962 NM_003752 LUA#82 1327 1015 949 1033 56 1119 1129.5 1088 467 1103 NM_018256 LUA#83 1250.5 951 1138 1055 66.5 1193 1204.5 1181 447.5 1205 NM_001948 LUA#84 2244.5 2685 2401.5 2620 110.5 2687 2842.5 2584 1071 2714 NM_005566 LUA#85 1709 1526.5 1628 1860.5 67.5 1881 1746.5 1895 630 1630 NM_021103 LUA#41 1926 2244.5 2051 2229 111.5 2407 2540 2141 1271 2148 NM_002970 LUA#42 624 821 659.5 791 125 970.5 975 816 233 695 NM_003332 LUA#43 1965 1940.5 1658 1865 312 2451 2442.5 2074 594 1769 NM_004106 LUA#44 347.5 348.5 281 335 53 351 392 399 96 302 NM_002982 LUA#45 2713 3642 2895 3096 129.5 3463.5 3752 3173 1511 3062 NM_005375 LUA#86 2159 2531 2256 2421 167.5 2822 2752 2748 1102 2492.5 NM_000250 LUA#87 2546.5 2107 2130.5 2120 88 2263 2364 2168 1006 2120 NM_004526 LUA#88 1386 1245 1195 1263 84 1418 1400.5 1287 493.5 1316.5 NM_004741 LUA#89 933 1599 1127.5 1113.5 153 1546 1679 1620 315.5 1160 NM_002467 LUA#90 1956 1673 1851 1710.5 295 2200 2298 1831 739.5 1677 ACTB LUA#91 2149.5 2840.5 3108 3160.5 93 3297 3543 3123 1706 3268 TFRC LUA#92 1049 775 707.5 768 73 1002.5 1062 878.5 259 904 GAPDH_5 LUA#93 1561.5 2061 2169.5 2073 80 2401 2387 2222 1175 2449 GAPDH_M LUA#94 2911.5 3948 3761 3945 135 4111 4218 3809 2710.5 4026 GAPDH_3 LUA#95 2910 4091 3621 4239.5 277 4336 4378.5 3889 3607 4420.5 Table 5F. Microtiter plates FlexMap description ID tretinoin10 tretinoin11 tretinoin12 tretinoin13 tretinoin14 tretinoin15 tretinoin16 tretinoin17 tretinoin18 tretinoin19 NM_005736 LUA#1 645 651.5 674.5 735.5 698 796 882 791 689 699 NM_000070 LUA#2 664 625 565 704 699 728.5 711.5 723.5 700 635 NM_018217 LUA#3 1364 1259 1292.5 1313 1384.5 1423 1521.5 1476 1348 1316 NM_004782 LUA#4 1107 1102 1041 1177.5 1148 1130 1235 1243 1214 1168 NM_014962 LUA#5 1169 1120 1197 1283 1243 1247.5 1277.5 1244 1215.5 1154 NM_004514 LUA#46 1104.5 1065.5 1095 1188.5 1228 1147 1236 1267 1212 1126.5 NM_006773 LUA#47 1012 1004.5 946 1062 1037 1097 1217.5 1141 1139.5 1101.5 NM_014288 LUA#48 777.5 770 765 771 805.5 793 896.5 895 861 801.5 NM_017440 LUA#49 557 520 523 557 591 626.5 692 633 588 568 NM_007331 LUA#50 881 812 753 849 919 879 897 978 854.5 837.5 NM_173823 LUA#6 963 952 995.5 1024.5 1050 1081 1034 1040 1026 946 NM_000962 LUA#7 762 738.5 738.5 864 902 752.5 845 860 784 779 NM_003825 LUA#8 299 334 341.5 358 252 310 327.5 324 309 274.5 NM_016061 LUA#9 1213 1145.5 1169.5 1280 1352 1241 1351 1380 1258 1197.5 NM_000153 LUA#10 156.5 142 135 150 148.5 138 166 175 157 144.5 NM_006948 LUA#51 69 62 63.5 72.5 65.5 66 72 80 64 62.5 NM_004631 LUA#52 768 722.5 723 823 790 782 743 734 715 668.5 NM_002358 LUA#53 472 428 395 462 455 552 548 527 445.5 414 NM_013402 LUA#54 1081.5 1089.5 1098 1196 1271 1266.5 1222 1215 1143 1087 NM_000875 LUA#55 1088.5 1079.5 1051 1151 1112 1167 1284 1241 1177 1060 NM_001974 LUA#11 169.5 194 254 355 223.5 263 231 205 211 175 NM_000632 LUA#12 393.5 405 394 451.5 442 478 551 484.5 434.5 403 NM_006457 LUA#13 74 71 80 75 77 84 82 75.5 77 67 NM_000698 LUA#14 190 205 169 233 215 234 237.5 218.5 214 184 NM_032571 LUA#15 194 177 178 217 217 219 212 217.5 198 208 NM_006138 LUA#56 412.5 383 396 459.5 498 441 511.5 528.5 429 436 NM_015201 LUA#57 1394 1432 1363 1500 1520 1702 1654.5 1623 1554 1485 NM_006985 LUA#58 1445 1332 1321.5 1558.5 1539.5 1511 1579 1614 1465 1349 NM_004095 LUA#59 677.5 673 714 723 761 813 888 849 713 743 NM_005914 LUA#60 2195 1712.5 1855 1767.5 1964 2001.5 2183 2217.5 1849 1801 NM_007282 LUA#16 3404 3317 3420 3720 3640 3628 3771 3742 3829 3740 NM_003644 LUA#17 382 379 360 396 403 384 420 424 420.5 395.5 NM_001498 LUA#18 1428 1422 1451 1542.5 1650 1646.5 1659 1643 1534 1547 NM_003172 LUA#19 3489.5 3393 3442.5 3630 3640 3407 3561.5 3713.5 3684 3729 NM_004723 LUA#20 1006 1055 941.5 1072 1070 1033.5 1103.5 1121 1101 1058 NM_014366 LUA#61 1858 1818.5 1815 1958 1893 1955 2124 2045 1954.5 1939 NM_003581 LUA#62 461 459 490 1104 560.5 575.5 784 492 442 292.5 NM_018115 LUA#63 3868 3688 3621.5 3997.5 4012 4038 4183 4186 3995.5 3973 NM_021974 LUA#64 2317 2285 2229 2359.5 2501 2395 2375 2577 2473 2448 NM_024045 LUA#65 751 715.5 652 803 823 922 958 891.5 766 704 NM_004079 LUA#21 3401 3346 3386 3649 3578.5 3621 3487 3722 3500 3466 NM_000414 LUA#22 849 886 876 922 959 923 991 997 1006 975.5 NM_001684 LUA#23 3100 3084 3141 3356.5 3190 3092 3330 3482 3482 3375 NM_003879 LUA#24 1887.5 1750.5 1837 1884.5 1982 2019 1981.5 2000 1908 1739 NM_002166 LUA#25 3185 2943.5 2941 3269 3091 2616 2919 3433 3462 2977 NM_005952 LUA#66 876 786 799 803.5 902 990 1022.5 960 878 811 NM_001034 LUA#67 493 529 561 721 515.5 682 767.5 716 677 449.5 NM_003132 LUA#68 347.5 309 330 344 404 351.5 355 349.5 350.5 323 NM_018164 LUA#69 193 163 225 324.5 196 284 359 269 277.5 175 NM_014573 LUA#70 193 198 204 268 232.5 272 262 277.5 256 187 NM_014333 LUA#26 1261 1234 1285 1432 1339 1336.5 1431.5 1414 1322.5 1274 NM_006432 LUA#27 589 537 543 644.5 587 652 654 625 592.5 501 NM_000433 LUA#28 519.5 465 439 478 544 475 576 543 515.5 491.5 NM_000147 LUA#29 231 253 261 242 286 280 269 256.5 266 242 NM_000584 LUA#30 268 331.5 415.5 491 316 397 371.5 438 375.5 271 NM_006452 LUA#71 1525.5 1457 1651 1599 1661 1895 1960 1641.5 1669 1592 NM_005915 LUA#72 442 446 457 449 507.5 501 489 502 463 469 NM_005980 LUA#73 94 88 90.5 85 94.5 97 114 91 95 93.5 NM_002539 LUA#74 793.5 759 750.5 783 845 953 975 909 783 795 NM_019058 LUA#75 2292.5 2282.5 2565 2388 2535.5 2484 2654.5 2545 2583 2289 NM_004152 LUA#31 630 607 706.5 700 697 751 782 717 646 677 NM_004602 LUA#32 102 102 113 124 98 205 540 400 104 138 NM_018890 LUA#33 2566.5 2827.5 3299 3828 3031.5 3314.5 3385 2517 3080.5 2211 NM_001101 LUA#34 2072 1968.5 2040.5 2060.5 2190 2259 2320 2389.5 2222 2133.5 NM_006019 LUA#35 336.5 316.5 346 403 354 404 367 363 348 346 NM_004134 LUA#76 952 989 1087 1135 1163 1017.5 1092 1083 1053.5 1056 NM_005008 LUA#77 826 834.5 886 963.5 996 949 884 937.5 914 857 NM_020117 LUA#78 2051 2083 2189 2086.5 2301 2289.5 2334 2460 2260 2288 NM_001469 LUA#79 433 407 554 697 555 594.5 461 448.5 502.5 369 NM_021203 LUA#80 345 387 409 387 426 427 412 427 416 381.5 NM_002624 LUA#36 273 266 280 324 295 328 304 291 286 271 NM_004759 LUA#37 122 120 127 128 144.5 135.5 147 132 124 134 NM_002664 LUA#38 847.5 834 832 873 937.5 902 875 929 902.5 944 NM_000211 LUA#39 1491 1458.5 1552 1507 1624 1206 1321 1614 1564.5 1554 NM_002468 LUA#40 259.5 253.5 269 284 279 315 376 349.5 262 279 NM_000884 LUA#81 784 800 844 868 921 887 940 932 895 849 NM_003752 LUA#82 952 992 998 943 993 1078 1145 1140 982.5 993 NM_018256 LUA#83 1089 1074.5 1091 1043 1123 1201.5 1267 1209.5 1127 1218 NM_001948 LUA#84 2343 2452 2481 2585.5 2510 2541 2508 2564.5 2473 2549 NM_005566 LUA#85 1548.5 1448 1550.5 1600 1689 1693.5 1735 1768 1561 1536.5 NM_021103 LUA#41 2065 1955.5 2142 2153 2196 2101 2263.5 2283 2152 2177 NM_002970 LUA#42 513.5 548.5 668 657 594 594.5 606 530 611 610.5 NM_003332 LUA#43 1333 1474 1892 1924 1823 1789 1557 1583.5 1724 1751.5 NM_004106 LUA#44 243.5 224 272 284 273 267 288 241 272.5 253 NM_002982 LUA#45 2470 2373.5 2700 2650 2725 2864 2951.5 2762 2734.5 2708 NM_005375 LUA#86 2294.5 2336.5 2443 2444.5 2426 2521 2719 2526.5 2478 2523.5 NM_000250 LUA#87 2043 1985 1993 2045 2198.5 2355.5 2463 2159 2135 2254 NM_004526 LUA#88 1153 1095.5 1178 1222.5 1322.5 1285 1299 1245 1257.5 1168.5 NM_004741 LUA#89 755.5 845 1009 1203 1030 1084 1146 1021 897 788 NM_002467 LUA#90 1510 1469 1648 1680 1767.5 1684 1670 1715.5 1649.5 1681 ACTB LUA#91 3243 3090 3181 3245 3443.5 3098.5 3174.5 3348 3385 3370 TFRC LUA#92 692 743 812 830 855 839 816 843.5 762.5 801 GAPDH_5 LUA#93 2242 1971.5 2105 2295 2386.5 2392 2183 2284 2124 2235 GAPDH_M LUA#94 3858 3566.5 3872 3913 3933 3983 3872 4090 3926 3949.5 GAPDH_3 LUA#95 3915 3968 4259 4355 4446 4043 4225.5 4362 4541 4571 Table 5G. Microtiter plates FlexMap description ID tretinoin20 tretinoin21 tretinoin22 tretinoin23 tretinoin24 tretinoin25 tretinoin26 tretinoin27 tretinoin28 tretinoin29 NM_005736 LUA#1 640 730 788 766 718.5 145.5 751 792.5 778.5 741.5 NM_000070 LUA#2 685 687.5 755 686 478 115 700 690 707.5 750 NM_018217 LUA#3 1359 1442 1484 1465 1196 235 1307 1438 1438 1492 NM_004782 LUA#4 1134 1250 1263 1217 962 226 1119 1230 1371 1286.5 NM_014962 LUA#5 1192 1211 1294 1182 872 218 1154 1277.5 1326.5 1336 NM_004514 LUA#46 1159 1194 1197.5 1151 971.5 243 1114 1193.5 1243 1193 NM_006773 LUA#47 1043 1086 1044.5 1145 874 229.5 1091 1167 1177.5 1171 NM_014288 LUA#48 867 887 849 859 606 198 905 883.5 896 853 NM_017440 LUA#49 563.5 606.5 635 668 504 126.5 572 648.5 629 633.5 NM_007331 LUA#50 804 879 877 868 608 128 875 901 875 857 NM_173823 LUA#6 1031.5 1028 1074.5 1021 710 89 1015.5 996 1138 1126 NM_000962 LUA#7 786 838 835 742.5 502 92 805 820 873.5 847 NM_003825 LUA#8 293.5 304 303.5 303 208 84.5 314 265.5 328 358.5 NM_016061 LUA#9 1161 1212 1243.5 1255 942 195.5 1273 1292 1309 1366 NM_000153 LUA#10 142.5 166 147 124.5 108 42 176 157 173.5 164 NM_006948 LUA#51 63.5 72 79.5 82 59 30.5 88 79 73 75.5 NM_004631 LUA#52 675 735.5 722 673 420 123 680.5 683 730 736 NM_002358 LUA#53 404 452 468 552 396 65 522 505 462 479.5 NM_013402 LUA#54 1170 1190.5 1234.5 1175 847 159 1146 1184.5 1207 1299 NM_000875 LUA#55 1106 1147 1164 1109.5 1133 244 1050 1207.5 1186.5 1240 NM_001974 LUA#11 192.5 244 328 229 131 41 187 206 326 280.5 NM_000632 LUA#12 416 462 441 466 399 62 417 427.5 472 485.5 NM_006457 LUA#13 71 77 88 76.5 62 29 91 70 77 88 NM_000698 LUA#14 184 221 218.5 240 183 51 234 217 231 250 NM_032571 LUA#15 197 206 211 210 146.5 39 207.5 199.5 237 225 NM_006138 LUA#56 439 465 463 492 400 76 505.5 508 481 455 NM_015201 LUA#57 1474 1697 1545.5 1613 1288 239 1501.5 1566.5 1693 1688 NM_006985 LUA#58 1404 1426.5 1391 1466 1025 152.5 1520 1559.5 1588.5 1516.5 NM_004095 LUA#59 781 826.5 724 833.5 508 80.5 750 786 836.5 825 NM_005914 LUA#60 1889 2185 2217 2583 1861 314 1944 2405 2103 2084.5 NM_007282 LUA#16 3819 3830 3905 3599 3355 1195 3365 3717 3937 3873 NM_003644 LUA#17 418 413 423 392.5 312.5 90.5 407 394 423 439.5 NM_001498 LUA#18 1596 1583 1644.5 1592.5 1126 194.5 1507 1592 1675 1729 NM_003172 LUA#19 3734 3740 3765 3485.5 3527 1393.5 3551 3869 3875.5 3546 NM_004723 LUA#20 1025 1135.5 1076 967 712.5 142 1098 1078 1129 1205.5 NM_014366 LUA#61 1866 1983.5 1990 1945 2046 505.5 1985 2063 2011 2041 NM_003581 LUA#62 349 459 725 486 433.5 59 330 467 542 538 NM_018115 LUA#63 4087.5 4059 3982 3939 3665 1204 4071 4113 4167 4218 NM_021974 LUA#64 2484 2467.5 2483 2350 1757 441 2372.5 2637 2567 2591 NM_024045 LUA#65 729.5 798 779.5 770 587 99 747 790 826 797 NM_004079 LUA#21 3552 3605.5 3495 3377 2652.5 688.5 3443.5 3592 3531 3582 NM_000414 LUA#22 941 1003 979 933 633 96.5 1010 1015 1053 1005 NM_001684 LUA#23 3316.5 3530 3511 3136.5 3092 1247 3338 3430.5 3590 3642.5 NM_003879 LUA#24 1848 2031 1977 1896.5 1645 342 1984 2060 2126.5 2034 NM_002166 LUA#25 3071 3382.5 3762.5 2832 2572 617 3016 3708 3766.5 3602.5 NM_005952 LUA#66 814.5 845 850.5 894.5 737 127 851.5 850.5 872 913 NM_001034 LUA#67 484 710 707.5 608 337 64 394.5 517 729.5 805 NM_003132 LUA#68 333.5 304 353 334 199 50.5 314 304.5 342 355.5 NM_018164 LUA#69 170 247 293 223 225.5 51 168 196 284 297 NM_014573 LUA#70 189 231 251 273.5 154 52 216.5 199 237 267.5 NM_014333 LUA#26 1265 1399.5 1470 1456.5 1091 254 1237 1396 1489.5 1499 NM_006432 LUA#27 554.5 605.5 690 686 480 101.5 574 628.5 679 694.5 NM_000433 LUA#28 541 499 552 488.5 308 69 478 551.5 545 548.5 NM_000147 LUA#29 273 266 299 278 198.5 49 207 257.5 286 277.5 NM_000584 LUA#30 231 358 433 352 199 57 314 280 411 431 NM_006452 LUA#71 1830 1544.5 1789 1876.5 1259.5 252 1497 1660 1647 1637 NM_005915 LUA#72 460.5 493.5 510 433.5 306 76 502.5 519 491 498 NM_005980 LUA#73 97 94 96 96 83.5 40 101.5 104 95 90 NM_002539 LUA#74 902 784.5 850 893 623 129 754 831 786 828 NM_019058 LUA#75 2347 2353 2439.5 2270 1909 415 2069.5 2436 2667 2983 NM_004152 LUA#31 679 718 764 752 450 88 610 631 712 768 NM_004602 LUA#32 95 108 161 115 685 100 321.5 251 106 111 NM_018890 LUA#33 2323 3545 3734 3090 3474.5 868 2600 2734.5 3577 3830 NM_001101 LUA#34 2216 2108.5 2276.5 2231.5 1848.5 439 2166.5 2230 2157.5 2312 NM_006019 LUA#35 388 349 414 328.5 242 56 344 357.5 392 417 NM_004134 LUA#76 1041 1069 1060.5 987 651 132 1092 1095 1138.5 1200 NM_005008 LUA#77 853 895 1077 907 520 118 791 852.5 942 939.5 NM_020117 LUA#78 2468 2232 2341 2280.5 1844.5 467 2069 2395 2201 2287 NM_001469 LUA#79 425 580 740.5 599 359 81.5 467 500.5 586 636 NM_021203 LUA#80 415 396 437 372 217.5 57 363 405 409.5 445 NM_002624 LUA#36 274 294 336 309.5 241.5 48 285 292 355 346 NM_004759 LUA#37 149 116 151 130 114 42 178 122 140 139 NM_002664 LUA#38 977.5 914 985 863 577.5 111.5 817 950 905 914 NM_000211 LUA#39 1765 1572.5 1714.5 1155 740 226 1502 1608 1601.5 1621 NM_002468 LUA#40 284 300 313 272 277 55 266.5 327 329 307.5 NM_000884 LUA#81 903 886.5 945 903 570 118 819.5 863 870 906 NM_003752 LUA#82 1041 1060 1104 1061 837.5 170 938.5 1068 1038 1035 NM_018256 LUA#83 1108 1137 1196 1134 827.5 167 1206.5 1275 1135 1244 NM_001948 LUA#84 2580 2584 2660 2423.5 1685.5 408 2252.5 2493 2630 2638 NM_005566 LUA#85 1561 1647 1700 1554 1127.5 209 1471 1537 1646 1690.5 NM_021103 LUA#41 2307 2172 2274 2057 1814 618 2016 2179 2230 2237.5 NM_002970 LUA#42 606 572 551 556 317 112 622 574 616.5 627 NM_003332 LUA#43 1942.5 1967.5 1914 1850 794.5 344 1313 1673 2015 2047 NM_004106 LUA#44 272 246 291 273 172 64.5 313 251 305 293 NM_002982 LUA#45 2717.5 2736 2784.5 2777 2331 489 2387.5 2700.5 2681.5 2746.5 NM_005375 LUA#86 2474 2594 2548 2496 1566.5 360 2233 2415 2634 2525 NM_000250 LUA#87 2214.5 2002.5 2248 2313 1710 399.5 1844 2084 2015 2121 NM_004526 LUA#88 1164 1159 1256 1110 796 197 1091 1190 1226 1219 NM_004741 LUA#89 883 948.5 1013.5 920 617 168 738 768 924 987 NM_002467 LUA#90 1628.5 1730 1731 1783 1174 317 1467 1738 1729.5 1861 ACTB LUA#91 3368 3386 3350.5 3125 2605 672 3275 3524.5 3469 3388 TFRC LUA#92 860.5 835 930.5 845 477 109 774 838 892 890 GAPDH_5 LUA#93 2317.5 2229 2328 2155 1768 404.5 2142 2223 2241 2203 GAPDH_M LUA#94 3957 3991 4132 3551.5 3745 1082 3577 4067 3950.5 3995 GAPDH_3 LUA#95 4351 4364 4325 4183.5 3891.5 2279 3800.5 4394 4434 4483 Table 5H. Microtiter plates FlexMap description ID tretinoin30 tretinoin31 tretinoin32 tretinoin33 tretinoin34 tretinoin35 tretinoin36 tretinoin37 tretinoin38 tretinoin39 NM_005736 LUA#1 769 747 1238.5 1115 813.5 721 962.5 1272 847.5 790 NM_000070 LUA#2 689.5 657 758.5 754 803 540.5 741 761 740.5 589.5 NM_018217 LUA#3 1322 1374 1573 1436.5 1463 1150 1445 1403 1383 1174.5 NM_004782 LUA#4 1185.5 1099 1239 1216.5 1215 921 1141.5 1079 1213.5 956 NM_014962 LUA#5 1150.5 1180 1240.5 1191 1253 853 1132 1133 1258 1011 NM_004514 LUA#46 1094.5 1087 1186 1169.5 1242 941 1167 1131 1193 977.5 NM_006773 LUA#47 1033 990 1208 1122 1156 847 1103.5 1060.5 1077 922 NM_014288 LUA#48 810 728 911.5 842 881.5 617 825.5 791 787 644 NM_017440 LUA#49 589 605.5 756 676 636.5 465 606 652 646 518.5 NM_007331 LUA#50 817 843 963 889 941 646.5 873 889.5 926 727 NM_173823 LUA#6 1059 1019.5 1053 1013.5 1122 648 994 985.5 1175.5 938 NM_000962 LUA#7 852 700.5 819.5 858 900 571.5 816 798 844.5 643 NM_003825 LUA#8 306 343 267 332 340 243 288 287 360 352 NM_016061 LUA#9 1193 1073.5 1220.5 1219 1260 928.5 1243.5 1193 1168 1021 NM_000153 LUA#10 142 139 155 171 143.5 110.5 152 145 173 140 NM_006948 LUA#51 76.5 77.5 91 84 92 68.5 75.5 79 78 74 NM_004631 LUA#52 698 714 696 645 717 415 656 645 773.5 621 NM_002358 LUA#53 461 581 624 570 530.5 354 470.5 458 553 484 NM_013402 LUA#54 1164 1118 1162 1205 1236 814 1171 1055 1330 987 NM_000875 LUA#55 1102 1155 1372 1375 1322 1146 1294 1286 1195 1045.5 NM_001974 LUA#11 305.5 276 191 246 259 162.5 192 206 295 226 NM_000632 LUA#12 441 482 621 688 451 309 426.5 453 450 447 NM_006457 LUA#13 73 106 88 88 92.5 59 79.5 84 96 98 NM_000698 LUA#14 261 264 271.5 260 282 176 237 269 279.5 260 NM_032571 LUA#15 201 224.5 213.5 200 216 141 223 213 231 214 NM_006138 LUA#56 460 420.5 539 534 484 367 443 470 470.5 449 NM_015201 LUA#57 1524 1740 1601 1563 1686 1294 1589.5 1575 1698 1441 NM_006985 LUA#58 1336 1304 1469 1513.5 1622.5 982 1517.5 1475 1453 1167.5 NM_004095 LUA#59 733 717 786 697 764.5 433 732 708.5 793.5 612 NM_005914 LUA#60 1856 2038.5 2571 2012.5 1880 1606 1870 1839 1896 1530 NM_007282 LUA#16 3508 3192.5 3565 3677 3822.5 3381 3679 3410 3550.5 2848 NM_003644 LUA#17 386.5 383 417.5 396 423 301 374 379 432 336 NM_001498 LUA#18 1574 1494 1634 1631 1704 1106 1567 1552 1756 1327 NM_003172 LUA#19 3400.5 3075 3448 3697 3747 3740 3828 3719 3455.5 2881 NM_004723 LUA#20 979 935.5 1064 1087 1182.5 753 1069 975.5 1032.5 798 NM_014366 LUA#61 1912 1786 2169.5 2125 2094 1938 2057 2071 1931 1729.5 NM_003581 LUA#62 580 836 497.5 667 676 406 453 544 633 625.5 NM_018115 LUA#63 3832.5 3397 4093 4088 4279.5 3877 4112.5 3898.5 3945 3394 NM_021974 LUA#64 2311 2023 2396 2418 2572 2001 2468 2388 2492 1925 NM_024045 LUA#65 778 821.5 869 774.5 847.5 557.5 752 697 801 700.5 NM_004079 LUA#21 3270 2953 3391 3470 3448 2730.5 3407.5 3299 3366 2635 NM_000414 LUA#22 997 865.5 1028 974 1061 630.5 996 885.5 948 778.5 NM_001684 LUA#23 3231 3000 3194.5 3326 3417 3351 3506.5 3213.5 3205 2689 NM_003879 LUA#24 1900 1735 2096 2056 2072 1621.5 2080 1945 1980.5 1569.5 NM_002166 LUA#25 2926 2752 2950 2945 2932 2688.5 2896 2989 2573 2189.5 NM_005952 LUA#66 843 867.5 978 977.5 953 645 842 796 927 766.5 NM_001034 LUA#67 718 878 591.5 781 904 488 618.5 586.5 694 572 NM_003132 LUA#68 342 274 322 361.5 318.5 210 307 316 370.5 250 NM_018164 LUA#69 315 320 241.5 360.5 357 178.5 201 241 354 292 NM_014573 LUA#70 252.5 283 251 305 286 220 233 257 280 232 NM_014333 LUA#26 1319 1406 1465 1494 1419 1092.5 1371.5 1338 1429.5 1161 NM_006432 LUA#27 661 705.5 664 722 683 461.5 592 634 716 572 NM_000433 LUA#28 523 441 538 528 568 326 499 457 537 346 NM_000147 LUA#29 258 300 313 275 294 195 304 253.5 326 246 NM_000584 LUA#30 391.5 493 276 416 451 259 288 399.5 432 343.5 NM_006452 LUA#71 1577.5 1658 1888 1709 1735 1113 1497 1516.5 1840 1397.5 NM_005915 LUA#72 481.5 510.5 523 583.5 576 356 504.5 415 564 392.5 NM_005980 LUA#73 90 99 124 123 112 81 100 99 105.5 108 NM_002539 LUA#74 830 864 906.5 944.5 939 599 833 759 913 662 NM_019058 LUA#75 2325 2239 2435 2754 2662 1983 2392.5 2181 2399.5 1863 NM_004152 LUA#31 707 677 734 995 718 405 615.5 675 744 547 NM_004602 LUA#32 153.5 214 909 1332 203 575 362.5 661 173 545 NM_018890 LUA#33 3389 3144 3168 4165 3486 2889 2506 3509 3510 2841.5 NM_001101 LUA#34 2091 2067 2357 2333 2374 1832 2294 2114.5 2236 1732 NM_006019 LUA#35 361.5 380 336.5 375 398 238 335 355 418 362 NM_004134 LUA#76 1049 803.5 933.5 1017 1043.5 699.5 1017 1031 1077.5 763 NM_005008 LUA#77 878 900 828 936.5 1033 581 886.5 860.5 949.5 736 NM_020117 LUA#78 2230 2093 2431 2387.5 2540.5 1912 2311 2187 2354 1724 NM_001469 LUA#79 691.5 645 471 654 680.5 506.5 464.5 577 767 627 NM_021203 LUA#80 400 353 369 437 476 210 385.5 351.5 442 327 NM_002624 LUA#36 312.5 357 327 291 357 234 326 320 376 332.5 NM_004759 LUA#37 147 125.5 134 177 156 107 133 132 158 121.5 NM_002664 LUA#38 867 808 927 916.5 1041 612 854.5 843 960.5 655 NM_000211 LUA#39 1459.5 1090 1164 1588 1684 1053 1460.5 1363 1622.5 766.5 NM_002468 LUA#40 270 364 428 462.5 356 285 336 540 393 353.5 NM_000884 LUA#81 847 824 971 938 986.5 575 838 813 932 759.5 NM_003752 LUA#82 968 1037 1171.5 1058 1106 797 1027.5 973 1082 863 NM_018256 LUA#83 1156.5 1089 1223 1153 1309 858 1084.5 996 1123 870 NM_001948 LUA#84 2417 2293.5 2372 2431 2615 1881.5 2387 2286 2542 1863 NM_005566 LUA#85 1603.5 1464.5 1570 1600.5 1792 1039 1520.5 1348 1667.5 1186.5 NM_021103 LUA#41 2062 1819 2376 2712 2331.5 1867.5 2178 2117 2183 1664 NM_002970 LUA#42 557 632 554 689 557 324.5 443 542 579 454 NM_003332 LUA#43 2024 1928 1382 1319.5 1562 875.5 1550.5 1649 2060 1620 NM_004106 LUA#44 259 257.5 287.5 297 295 172 238 298 282 235 NM_002982 LUA#45 2586 2449 3029.5 3071 2895 2314 2978 3409 2749.5 2503.5 NM_005375 LUA#86 2374.5 2331 2476 2278.5 2453.5 1650 2201 2224 2486 2014 NM_000250 LUA#87 2007 1994 2529 2190 2281 1760 2035 2053.5 2251 1773 NM_004526 LUA#88 1199.5 1072 1200 1291 1302 863 1242 1132.5 1240 874 NM_004741 LUA#89 948 938 694 1441 960 494 724 909 941 817 NM_002467 LUA#90 1735 1597 1675.5 1910.5 1668 1121 1574.5 1764.5 1806 1401 ACTB LUA#91 3074 2741 3236 3178 3290 2654.5 3268 3235 3265 2408.5 TFRC LUA#92 899 882 882 800.5 940 534 833 845 1049.5 774.5 GAPDH_5 LUA#93 2041.5 1918 2170.5 2325 2409.5 1721 2170.5 2079 2197 1628 GAPDH_M LUA#94 3615 3383 3924 4094 4111 3702 4060 3901 3849 3216 GAPDH_3 LUA#95 4065 3741 4295 4166 4220 4140 4339.5 4356 4121 3415 Table 5I. Microtiter plates FlexMap description ID tretinoin40 tretinoin41 tretinoin42 tretinoin43 tretinoin44 tretinoin45 tretinoin46 tretinoin47 NM_005736 LUA#1 92 522 909 1432 1896 847 1205.5 695 NM_000070 LUA#2 84.5 400 756 769.5 717.5 638.5 741 490.5 NM_018217 LUA#3 189 990 1514 1581 1499 1286 1386 854.5 NM_004782 LUA#4 163 811 1306 1264 1063.5 1077 1094 639 NM_014962 LUA#5 152.5 726 1269 1290 1213 1076 1134 717 NM_004514 LUA#46 176 834 1159.5 1224 964 992 1017 626 NM_006773 LUA#47 164 717 1137 1120.5 783 937.5 907 576.5 NM_014288 LUA#48 154 477.5 801 816 527.5 706 650 453 NM_017440 LUA#49 101 405.5 707 774 716 607 693 477 NM_007331 LUA#50 88 474 915.5 927.5 770 739 835 583 NM_173823 LUA#6 84 594.5 1106.5 1168.5 1130.5 1080 1163 807 NM_000962 LUA#7 63.5 391 761 776 470 646.5 671.5 452 NM_003825 LUA#8 81.5 192.5 338 383.5 307.5 331 502 476.5 NM_016061 LUA#9 134 674 1086 1267.5 863 975 1073.5 741 NM_000153 LUA#10 35 90 138 150 120 138 171.5 223 NM_006948 LUA#51 34 49 82 95 83 79.5 85.5 106.5 NM_004631 LUA#52 86.5 322 705 629 524 633 667 532 NM_002358 LUA#53 64 339 572 611 366 481 531 433 NM_013402 LUA#54 114 705 1150 1150 806 1051.5 1087 711 NM_000875 LUA#55 184 1002 1389.5 1772 1303 1192 1271 810 NM_001974 LUA#11 38.5 98 351 256 258 253.5 286.5 224 NM_000632 LUA#12 60 298 544 994 932 486 500 525.5 NM_006457 LUA#13 36 51 83 94 84 95 132 200 NM_000698 LUA#14 45.5 156 269 394 342 297.5 363 352 NM_032571 LUA#15 31 109 203 222.5 165.5 191 270.5 253.5 NM_006138 LUA#56 70 325.5 443 659.5 488 437 429 341 NM_015201 LUA#57 182 1154 1768 1714 1251 1398.5 1507 930.5 NM_006985 LUA#58 90 720 1223 1310 813 1136.5 983 635 NM_004095 LUA#59 72 383 714 665 496.5 650.5 593.5 442 NM_005914 LUA#60 303 1363 2538.5 2273 1739 1694 1933.5 1154.5 NM_007282 LUA#16 778.5 3067.5 3626 3678 3097 3055 2958.5 1505 NM_003644 LUA#17 69 255 415 428 359 374 395 302 NM_001498 LUA#18 126.5 890.5 1632 1563.5 1134 1467 1510 888 NM_003172 LUA#19 818 3200.5 3348 3577.5 2983 2898 2747 1471 NM_004723 LUA#20 89 620 1056 982 761 886 853 494.5 NM_014366 LUA#61 383 1773 2236 2380 1850 1787.5 1681.5 1009.5 NM_003581 LUA#62 62.5 372 962 808 751 735.5 801 515 NM_018115 LUA#63 746.5 3193 3722 4141 3225.5 3292.5 3054 1569 NM_021974 LUA#64 268 1606 2307 2301 1472 1996 1890 1117.5 NM_024045 LUA#65 85 495 831 833.5 527 681 728.5 471 NM_004079 LUA#21 350 2202 3008 3030.5 2326 2816 2701 1488 NM_000414 LUA#22 79.5 477 902 920 503 800 838 646 NM_001684 LUA#23 884 3012 3316.5 3512 3036 2755.5 2662 1478 NM_003879 LUA#24 225 1355.5 1937.5 1983.5 1275 1677 1564 923 NM_002166 LUA#25 411.5 1693.5 2999 2964 2068 1985 2518 1239 NM_005952 LUA#66 104 573 891 961 623.5 847.5 775 571 NM_001034 LUA#67 65 447 989 779 733.5 645 1063 476 NM_003132 LUA#68 41 126 264.5 264 195.5 257 296 327.5 NM_018164 LUA#69 52 170 429 380 304.5 304 358.5 234 NM_014573 LUA#70 43 143 320 325 274 243 407.5 300 NM_014333 LUA#26 180.5 949.5 1577 1549 1309 1337 1390 811 NM_006432 LUA#27 90 394 854.5 800 695 670 719 450 NM_000433 LUA#28 53 234 451.5 466 318 421 370.5 244.5 NM_000147 LUA#29 48 184 276.5 330.5 274 275 318 288 NM_000584 LUA#30 45 189 470 461 435 379 597 352 NM_006452 LUA#71 207.5 1176 1736 1656.5 1374 1531 1587 911.5 NM_005915 LUA#72 48 300 495 474 315.5 496 401.5 256.5 NM_005980 LUA#73 35.5 83 102 162 149 95 114 168 NM_002539 LUA#74 94 559 886 952 715 861.5 822 535 NM_019058 LUA#75 258 1804 2807 2882 2288 2430 2201 1184 NM_004152 LUA#31 58 337 710 811 628.5 782 652 408.5 NM_004602 LUA#32 458.5 678 736.5 1957 1765.5 664 571.5 773.5 NM_018890 LUA#33 562 2557 3812.5 4178 4310 3724.5 2984 1462.5 NM_001101 LUA#34 260.5 1606 2206 2257 1647 1943 1881 904 NM_006019 LUA#35 41 192.5 363 400.5 353 361 402 332 NM_004134 LUA#76 78 432.5 873 866 531 759 744 493 NM_005008 LUA#77 73 425 885 923 779.5 801 828 525 NM_020117 LUA#78 236.5 1585 2193 2242.5 1690 1988 1734 848.5 NM_001469 LUA#79 65 320.5 885 739 765 734 561 370 NM_021203 LUA#80 53 171 386 377.5 283.5 327 348 279 NM_002624 LUA#36 53 180.5 397 381 330.5 336 400.5 394 NM_004759 LUA#37 40.5 67.5 186.5 169 134.5 142 145 190 NM_002664 LUA#38 73 478 906 798 599 773 748 512 NM_000211 LUA#39 81 598.5 1229 1162.5 868 1061 973 440.5 NM_002468 LUA#40 48 285.5 324 728 896.5 343 632 464 NM_000884 LUA#81 82 506 875 897 732.5 789 805 513.5 NM_003752 LUA#82 115.5 536.5 1061 1015.5 768.5 1020 912.5 596 NM_018256 LUA#83 102 717 1252 1104 692.5 1077 929 470 NM_001948 LUA#84 255 1430 2470.5 2273.5 1891 2131 2132.5 1104 NM_005566 LUA#85 129.5 862.5 1842.5 1550 1000 1282 1196 592 NM_021103 LUA#41 591.5 1730 2238 2731 2283 1975 1828.5 1027 NM_002970 LUA#42 92 298 598 589 622.5 577.5 586 478 NM_003332 LUA#43 274 646 1341.5 1324 1984.5 1651 2226 1241.5 NM_004106 LUA#44 47 142 253.5 302 321 271 273.5 267 NM_002982 LUA#45 286 2331 2514 4037 4231 2722 2898 1696.5 NM_005375 LUA#86 273 1380 2455 2357.5 2068 2193 2264 1273.5 NM_000250 LUA#87 261.5 1504 2135 2243 1820 2094 1863 1124 NM_004526 LUA#88 108 642 1081.5 1122 840 1033 1012 641.5 NM_004741 LUA#89 131 383 972 1003 933 920 1126 571 NM_002467 LUA#90 383 983.5 1643 1800 1920 1518 1725 1034 ACTB LUA#91 344 2113 2976 3020 2200 2601 2521 1195 TFRC LUA#92 89 400 794 910.5 811 827.5 1042 592 GAPDH_5 LUA#93 238 1501 2205.5 2064 1482 1768.5 1737 843 GAPDH_M LUA#94 642 3318 3783.5 3886 3205 3303 3248 1513 GAPDH_3 LUA#95 1659 3754 4065 4240 3880 3379 3353.5 1707.5

TABLE 6 A Experiment 1- Blank and DMSO description FlexMap ID BLANK BLANK DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO NM_005736 LUA#1 15 30 232 237 270.5 227.5 243 224 230 261 275.5 258 NM_000070 LUA#2 23.5 30 234.5 198 193 219.5 197.5 187 203 225.5 242.5 234 NM_018217 LUA#3 16 21 510 513 510 505 507.5 458 490 523 534 530 NM_004782 LUA#4 34.5 31 449.5 592.5 581 603 605 552 606 605 615.5 608.5 NM_014962 LUA#5 26.5 28 318.5 457 473 482.5 486 438 467 456 500 460 NM_004514 LUA#46 29 35.5 553 424 419 452.5 436 394 449 509 477 470.5 NM_006773 LUA#47 30 38 252 308 313 339 338 285 325 323.5 326 335 NM_014288 LUA#48 31 37.5 203.5 138 132.5 137 136 125.5 138 141 143 137 NM_017440 LUA#49 34 30 106 98.5 105.5 110 118 94 107 121 116 117 NM_007331 LUA#50 19 24 187 130 120 134 128.5 121 140 150 149.5 138 NM_173823 LUA#6 33 28.5 428 500 495 500.5 533 460 522 544 505 517.5 NM_000962 LUA#7 29 39.5 425 368.5 370 383.5 376 339 395 423 419 404 NM_003825 LUA#8 32 26 500.5 352 327 357 355 311 369 381 376 370.5 NM_016061 LUA#9 28 27 261 224 217 222 223 203 234 250 237 237.5 NM_000153 LUA#10 20 32.5 287 213.5 203.5 213 213 183 221.5 244 231 226 NM_006948 LUA#51 31 34 588 600 609.5 609.5 623 565 621.5 647 629 659.5 NM_004631 LUA#52 22 12 291 268 269 284.5 285.5 261 274 297 287 281.5 NM_002358 LUA#53 29 33 343 355 354.5 386 378 328.5 361 387 397 374 NM_013402 LUA#54 24 33 291.5 283 276 301 284.5 248.5 281 282 301 298 NM_000875 LUA#55 25 24 51 60 56 65 64 52.5 58.5 60.5 57 66 NM_001974 LUA#11 28 37 98 105 101 104 113 96 109.5 104 108 109.5 NM_000632 LUA#12 24 29 84 55.5 63.5 56 66 55 55 59 53 66 NM_006457 LUA#13 32.5 36 110 117 124 126 145 118 133 115 134 143 NM_000698 LUA#14 28 30 375.5 380.5 398 392.5 379.5 357 401 372 411 385 NM_032571 LUA#15 23 32 25 28 35 34 27 30 33 37 36 31.5 NM_006138 LUA#56 25 33 986.5 1084 1076 1125 1116.5 986 1104 1154 1109 1139 NM_015201 LUA#57 28 29 772 752 787 792 735 698 745 806 840 793 NM_006985 LUA#58 37 37 171 130 135.5 134 134 116 127 129 131.5 139 NM_004095 LUA#59 46 35 1656 1443.5 1428 1459 1379 1264 1389 1369.5 1487.5 1530 NM_005914 LUA#60 39 28 1214 1110 1128 1193 1211.5 1044.5 1117 1091 1211 1243 NM_007282 LUA#16 22 26.5 50 49 45 53 54 42 53 47.5 53.5 60 NM_003644 LUA#17 36.5 35 226 231.5 232 255 246.5 238.5 260 243 240 233.5 NM_001498 LUA#18 26.5 24 401.5 209 205.5 211 210 173.5 207 236 229.5 214 NM_003172 LUA#19 20 31 259 231 229 249 245 200 246 242 253 260 NM_004723 LUA#20 29 28 598 410 414 404.5 420.5 329.5 372.5 382 421 452 NM_014366 LUA#61 41 34 705 625.5 632 653 617 582 643 655.5 677 661 NM_003581 LUA#62 21 32.5 278 50 115 61 64 48 58.5 60 56.5 53.5 NM_018115 LUA#63 32 27 601.5 675 694 689 724 574.5 665 645 735.5 787 NM_021974 LUA#64 34.5 33 1652 1660 1680.5 1724 1666 1479 1664 1617 1804 1849 NM_024045 LUA#65 34 28 262.5 235.5 241 247 242 208 231 242 253 252 NM_004079 LUA#21 33.5 28 73 65 73 71 67 54.5 71 62 63 73 NM_000414 LUA#22 37.5 24.5 222 134 144.5 152 143.5 124 136.5 142 147 138 NM_001684 LUA#23 20 32 39 38 34 49 43 37 44 46 45 45 NM_003879 LUA#24 39 28 51 46 56 53 58 45.5 54.5 56 54 56 NM_002166 LUA#25 29.5 32 60 66.5 82 81 76 70 74 75.5 75.5 79.5 NM_005952 LUA#66 29 40.5 534.5 534 573 602.5 553 529 592.5 619 556 570 NM_001034 LUA#67 24 21 552 584 586 586 599 530 603 644 604 601 NM_003132 LUA#68 32.5 29 1555 1730 1763 1807 1782.5 1645 1830 1833 1824.5 1844.5 NM_018164 LUA#69 29 28 428.5 431 425 418 411.5 361 433 438 462 499 NM_014573 LUA#70 41 44 589 360 361.5 387 383 338 399.5 419 400.5 397 NM_014333 LUA#26 23 29 69 69 85.5 84 85 65.5 77 82 83 82 NM_006432 LUA#27 25 31 312 272 276 294 275 247 270 306 302 290 NM_000433 LUA#28 30 22.5 252 142 135 135 138 120 141 153 146 160 NM_000147 LUA#29 34 25 102 101 102 106.5 106 84 97 102.5 106 100 NM_000584 LUA#30 30.5 31 1070 726 741 743 750 661 757 785 777.5 788 NM_006452 LUA#71 41.5 42.5 147.5 108.5 115 115 114.5 106 120.5 125 111 110 NM_005915 LUA#72 27 30.5 159.5 116 112 117 118 102 113 121 125 123.5 NM_005980 LUA#73 29.5 39 1277 1452 1399.5 1493 1439 1372 1425 1473 1473 1479 NM_002539 LUA#74 34 32 1594.5 1793 1769 1801 1828 1620 1725 1827 1992 1916 NM_019058 LUA#75 38 39.5 1044.5 886.5 872 897 876.5 792 830 814.5 946 930 NM_004152 LUA#31 26 28.5 1525 1952.5 2027 1926 2057 1856 1823 1940 1987 2025 NM_004602 LUA#32 34 28 195.5 192 193 200 203 178 200 203.5 204.5 198 NM_018890 LUA#33 40 39.5 771.5 596.5 617 647 633 592.5 692.5 700 684 645 NM_001101 LUA#34 31 27 1771.5 1972.5 1931 2061 1922 1789 1912 2051 2122.5 2118.5 NM_006019 LUA#35 38 22 514 534 509 553 526 486 567 589 577 552 NM_004134 LUA#76 33 32 955 610.5 597 619 626 576 611 646 607 605.5 NM_005008 LUA#77 36 51 962 911 889 908.5 906 806 874 855.5 958.5 916 NM_020117 LUA#78 31 35 1235.5 1359 1327 1435.5 1350.5 1243 1362 1424 1399.5 1404.5 NM_001469 LUA#79 39.5 40 1511 1917 1890 1972.5 1994.5 1780.5 1858 1848 1988 2024 NM_021203 LUA#80 41 42 1421.5 1578 1531.5 1552 1535.5 1367 1535 1558 1637 1653 NM_002624 LUA#36 33 26.5 1100 1042 1019.5 1048 1005 957.5 1035 1063 1020 1055 NM_004759 LUA#37 35 39 70.5 84 70.5 73 75 58 71 72.5 62 131 NM_002664 LUA#38 29 25 1467 1319 1313.5 1370 1303.5 1184 1326.5 1428 1394 1398 NM_000211 LUA#39 36 33.5 932 663 621.5 675 660 612.5 679 699 702 686 NM_002468 LUA#40 23 25 134.5 130 139 152 143.5 131.5 143.5 144 147 152 NM_000884 LUA#81 40 46 1284 1582 1611 1668 1647 1514 1652 1669 1670 1688 NM_003752 LUA#82 41 46 216 245 255 244 249 219 230.5 266.5 244 254.5 NM_018256 LUA#83 31.5 28.5 665.5 1012 1090 1120 1141.5 1160 1115 953.5 1044 1014 NM_001948 LUA#84 34 27 180.5 155.5 156 157 154 137 150 168.5 161 159 NM_005566 LUA#85 41 34 2231 2060 2128.5 2169 2106.5 1939.5 2064.5 2145 2116.5 2100 NM_021103 LUA#41 34.5 30 1272 1437 1473 1503 1443 1414 1506 1456 1501 1461 NM_002970 LUA#42 41 24.5 396 450.5 462 478 479 430.5 496 471.5 480 481 NM_003332 LUA#43 34.5 35 838.5 1008 1029.5 982.5 978 931 1004 1061 1030 1037 NM_004106 LUA#44 27.5 30 296 278 282.5 302.5 282.5 276 306 324 291 291 NM_002982 LUA#45 25.5 32 504 487 513 542 502 467 524 578.5 529 521.5 NM_005375 LUA#86 46 38 1101.5 1745 1753 1785 1811 1641 1712.5 1731 1726 1662.5 NM_000250 LUA#87 39 37 2256 2007.5 2043 2043 2031.5 1774 1918.5 1971 2128 2032.5 NM_004526 LUA#88 37 29 853 854 851 889 854 770 833 834.5 872 873 NM_004741 LUA#89 40 36.5 484.5 567 584 610 603 564 622 652 598 554.5 NM_002467 LUA#90 44 52 1411.5 2347 2409 2476 2397.5 2416 2415.5 2431 2435 2296 ACTB LUA#91 40 39 1480 1420 1437 1536.5 1514 1336 1470 1606 1527 1524.5 TFRC LUA#92 49 55 508 556 585 587.5 578.5 519 572 623 603 591 GAPDH_5 LUA#93 55 57.5 1707 2319.5 2460 2510 2602 2496 2654 2758.5 2441.5 2356 GAPDH_M LUA#94 51 29 2351 2607 2800 2937 2802 2679 2809 2793 2767 2698 GAPDH_3 LUA#95 53 47 2550 3645 3798 3870 3894 3590.5 3663.5 3824 3859 3782 B Experiment 1- Tretinoin FlexMap description ID Tretinoin Tretinoin Tretinoin Tretinoin Tretinoin Tretinoin Tretinoin Tretinoin Tretinoin Tretinoin NM_005736 LUA#1 319 351 89 329 319.5 138.5 309 279 308 336 NM_000070 LUA#2 269.5 370 104.5 354 372 159 336 318 329.5 386 NM_018217 LUA#3 659.5 824 225.5 823 800 343 785 727 747 886 NM_004782 LUA#4 635 855 232 870 812.5 341 855 750 790.5 907 NM_014962 LUA#5 414.5 556 171.5 552.5 567 232 576.5 516.5 537 575.5 NM_004514 LUA#46 262 320 96 306 313 144.5 302 286 312.5 356 NM_006773 LUA#47 139 213 56.5 216 219 79.5 235 174 213 257 NM_014288 LUA#48 55 61 45 60 56 41.5 60 59 64 62 NM_017440 LUA#49 55 68 40 67 68 48.5 66.5 62 59 66 NM_007331 LUA#50 70 81 42 96 87 53 91 80 85.5 97 NM_173823 LUA#6 482.5 654 186.5 675 646 291 604.5 573 591.5 718 NM_000962 LUA#7 663 823 294 793.5 807 398 772 764 754 894 NM_003825 LUA#8 476.5 630.5 256 580 609 318 584 550 557 658 NM_016061 LUA#9 297 401 128 385 382 173 357.5 357 362.5 422 NM_000153 LUA#10 324 419.5 120 384 408 170 374 377.5 357.5 432 NM_006948 LUA#51 592.5 791.5 224 773.5 795 325 762 716.5 742 840.5 NM_004631 LUA#52 238 334 103 331 310.5 135.5 326 289.5 304 352 NM_002358 LUA#53 72 98 53 96 99 58 98 85 99 113 NM_013402 LUA#54 62 75 43 72 75.5 48.5 75 71.5 72 71 NM_000875 LUA#55 53 62 36 62 65 48 74 54 63 68 NM_001974 LUA#11 288 435.5 95 414 430 141 420.5 396 403 466 NM_000632 LUA#12 222 292 78 307 277 114 275.5 251 254 323.5 NM_006457 LUA#13 113 135 78 123 136 90 151.5 132 144 135 NM_000698 LUA#14 1332.5 1743 503 1688 1686.5 787 1606 1552.5 1562 1779 NM_032571 LUA#15 125 161 60 169 164.5 81 172.5 146 144 188 NM_006138 LUA#56 93 124 51.5 113.5 115.5 66 122 114.5 114 130 NM_015201 LUA#57 139 222 64 208 203 87 194.5 183 181 226.5 NM_006985 LUA#58 47 59 37 55 56 40 57 51 52 54 NM_004095 LUA#59 148 227 78 212 212 101 214.5 194.5 203 247 NM_005914 LUA#60 566.5 843 209 866 848.5 351 885 795 881.5 948 NM_007282 LUA#16 39 64.5 42.5 64 62 56 61 64.5 60.5 60 NM_003644 LUA#17 195.5 252 105 266.5 259 142 271.5 260 282 272 NM_001498 LUA#18 279 402 96 374 412 140 360 335.5 361 424 NM_003172 LUA#19 208 286 86 264 257 121 260 235 243 276 NM_004723 LUA#20 318 394 127 418.5 388.5 184 400 363.5 380 446 NM_014366 LUA#61 163 235 66 231 235 97 231 209.5 213 263 NM_003581 LUA#62 147 80 50 65 52 43 50 42 53 57 NM_018115 LUA#63 552.5 735.5 143 690 601 227.5 582 418 506.5 644 NM_021974 LUA#64 872 1105 293.5 1102.5 1052.5 477.5 1068 955 1003 1158 NM_024045 LUA#65 100 145 52 141 142 69 133 119 124 146 NM_004079 LUA#21 93 124.5 55 127 122 70 125.5 99.5 113 129 NM_000414 LUA#22 270 442 100 408.5 415 145 402.5 373 372.5 470.5 NM_001684 LUA#23 54 66.5 41 65 65 43 61 63 69 68 NM_003879 LUA#24 57 80 41 71 76 53 80 67 72.5 77.5 NM_002166 LUA#25 124.5 159.5 61 168 159 79.5 156.5 152 154 180 NM_005952 LUA#66 149 198.5 72.5 189 203 95.5 188.5 182 172 212 NM_001034 LUA#67 157 225 73 209 212.5 92 219 185 191 243 NM_003132 LUA#68 410 540 148 523 517 212 488 467.5 488 596 NM_018164 LUA#69 131 165 57 152.5 155 75 143 142 140.5 177 NM_014573 LUA#70 99 153.5 61 138 155 79 138.5 144.5 135 155 NM_014333 LUA#26 366.5 531 134 492 530 197.5 497 459.5 472 584 NM_006432 LUA#27 1081.5 1409.5 397 1446 1405 625 1345 1203 1294.5 1495 NM_000433 LUA#28 442.5 640 144 605 622 228.5 564 532 536.5 647 NM_000147 LUA#29 573 861 195.5 822.5 850 302 783 763 764 894 NM_000584 LUA#30 1464 1938.5 476 1981.5 1945 799.5 1938 1717 1765 2115 NM_006452 LUA#71 67.5 79.5 41 75 68 53.5 82 74 78.5 76 NM_005915 LUA#72 39 54 34.5 44 56 41 51 47 44 59 NM_005980 LUA#73 106 163 57 142 163 74 149.5 151 145 173 NM_002539 LUA#74 231 326 85 313 314.5 131 300 281.5 276 362 NM_019058 LUA#75 143 164 59 158 152 79.5 148.5 129 143.5 158 NM_004152 LUA#31 1662 2073 775 2127.5 2117 1110 2045 1823 1944 2194 NM_004602 LUA#32 182 239 88 232.5 227.5 113.5 224 212 215.5 258 NM_018890 LUA#33 537.5 758 187.5 788 743.5 293.5 719 712 705 824 NM_001101 LUA#34 2773 2969.5 1490 2968.5 2890 1977 2893.5 2694 2722 3119.5 NM_006019 LUA#35 569 818 186 828 762 287 767 734.5 746 867 NM_004134 LUA#76 207 277 83.5 292 306 111 280 266.5 278 318 NM_005008 LUA#77 307 392 123 401 382.5 167 372 338.5 343 416 NM_020117 LUA#78 408 584.5 145 554 564 230 529.5 519 527 607 NM_001469 LUA#79 809 1179 284 1191 1179 463 1140.5 1077 1076 1221 NM_021203 LUA#80 442.5 642.5 151 578.5 611 228.5 563 546 547 654 NM_002624 LUA#36 1267 1418 576 1447 1402 820 1369 1224.5 1288 1492.5 NM_004759 LUA#37 148 139.5 53 128 141 67 134 103 116 149.5 NM_002664 LUA#38 2157 2552 892 2527 2504 1337 2394 2330 2325 2761 NM_000211 LUA#39 1125 1420 454.5 1349 1366.5 682 1361 1315 1294.5 1488 NM_002468 LUA#40 325 448.5 121 496.5 473 174.5 426.5 399.5 418 492 NM_000884 LUA#81 676 871.5 250 871 865 389 830.5 799 799 946 NM_003752 LUA#82 114 144.5 71 128.5 142 94 137.5 124.5 130.5 145.5 NM_018256 LUA#83 897 726 388 903 998 589 1256.5 1208 1557 747 NM_001948 LUA#84 61 73 47 63 71 51 76 66 58 75 NM_005566 LUA#85 583.5 642 150 607 596 219 577 523.5 540 632.5 NM_021103 LUA#41 2257.5 2689 925 2668.5 2611 1370 2590 2454 2412 2719 NM_002970 LUA#42 1181 1595 400 1478 1584 651 1503 1459 1455 1683.5 NM_003332 LUA#43 2219 2571.5 1100 2688.5 2573.5 1470 2528 2325 2387.5 2641 NM_004106 LUA#44 994 1303 373 1308 1315 576.5 1274.5 1218 1187 1452 NM_002982 LUA#45 3231 3797 1738 3852 3752 2466 3667 3451 3488 3786 NM_005375 LUA#86 523 594.5 238 631.5 617 351 734 717 780.5 638 NM_000250 LUA#87 137 194 74 177 187 95.5 173 166 164 198 NM_004526 LUA#88 150 213 77 208 192.5 101 206.5 182.5 192 223 NM_004741 LUA#89 168 215 100 204 198 116 214 204.5 205 208 NM_002467 LUA#90 702 736 256 792.5 842.5 399 957 976 1030 801 ACTB LUA#91 1929 2483 818 2425.5 2563 1173 2347 2296.5 2313 2609 TFRC LUA#92 191.5 285 81.5 280.5 289 109 272 251.5 250 305 GAPDH_5 LUA#93 998.5 1419 378.5 1433 1505.5 632 1469 1347 1299 1629 GAPDH_M LUA#94 1134 1511.5 460 1520 1502 698.5 1462 1334 1360.5 1575 GAPDH_3 LUA#95 2428 2979 1102.5 2911 2912 1645 2823 2559 2580 2982

TABLE 7 A Experiment 2- Blank and DMSO FlexMap description ID BLANK BLANK DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO NM_005736 LUA#1 30 38 48 53 245 256 258.5 259 226 275 219 208 NM_000070 LUA#2 31 26.5 39 39 198 207 202 235 180.5 201 193 202 NM_018217 LUA#3 34 26 50.5 94 550 605 604 639.5 531 629.5 544 531 NM_004782 LUA#4 36.5 37 46 85.5 600.5 569 593 654.5 556 689 629.5 538 NM_014962 LUA#5 39 36.5 50 74 486 492 469 496.5 415 590.5 469 411 NM_004514 LUA#46 29 29.5 39 90 562.5 607 641 633 497.5 539 597.5 605 NM_006773 LUA#47 26 27.5 29 60 489 496 528 572 475 479 499 491 NM_014288 LUA#48 23 26 27 35 171 170 154 163.5 138.5 158 161 145 NM_017440 LUA#49 35 32 26 36 135 144 129 159 128 134 146 141 NM_007331 LUA#50 31 23 25 31 148 161.5 182.5 182 119 149.5 150 150 NM_173823 LUA#6 18 20 42 71 502 447 463 504 432 573.5 501 462 NM_000962 LUA#7 25 25 59 91 401 406 398 403 330 411 397 371.5 NM_003825 LUA#8 26.5 34 101 114 433 423.5 419 420.5 352.5 418.5 400 405 NM_016061 LUA#9 21.5 23 41 60 256.5 250.5 257 268 222 275 243.5 233 NM_000153 LUA#10 29 30 38 47.5 250 268 260 264 226 264 243.5 229 NM_006948 LUA#51 28.5 41 51 100 708 696 668 684 579 724 667 631 NM_004631 LUA#52 32.5 35 37 49 308 320 323 328 280 335 309 307 NM_002358 LUA#53 30 33 35 42 431 395 435 419 362 429 418 401 NM_013402 LUA#54 23 28 32 56.5 349 342 360 380 313.5 353 340 349 NM_000875 LUA#55 20 28.5 27 27 85 90 92 110 105 90.5 93 79 NM_001974 LUA#11 19 30 24 27 129 146 121.5 125 94 139 102 102 NM_000632 LUA#12 20 33.5 24 26 72 80 82 82 76 72 67 81 NM_006457 LUA#13 33 35 49 51 140 153 132 152 126 143 118.5 92 NM_000698 LUA#14 30 27.5 47 80 467 500 484 483 398 475 451 418 NM_032571 LUA#15 25 23 22 21 21 30 26 22 32.5 29 29 23 NM_006138 LUA#56 36 29 71 200 1270 1262 1328.5 1397 1193 1225 1253 1285 NM_015201 LUA#57 26 30 45 117.5 849.5 896 938.5 929 725 845 846 878 NM_006985 LUA#58 26 33 33 34 146 144 144.5 133 111 145 147 111 NM_004095 LUA#59 31 38 115 311 1642 1798 1731 1809 1469 1644 1613 1462.5 NM_005914 LUA#60 32 24 71.5 218 1471 1443 1509 1635.5 1124 1420.5 1406.5 1263 NM_007282 LUA#16 27 35 24.5 20 42.5 46 43 46 45 44 46 40 NM_003644 LUA#17 30 34 49 53.5 252 221 229 232 192 223 198 217 NM_001498 LUA#18 22 35 27 37 236 268 276.5 300 252 263 258 266 NM_003172 LUA#19 33 27 30 43 257 266 270 268 218 276.5 245 240 NM_004723 LUA#20 30 17 45 90 536 581 535 621 482.5 569 496 439 NM_014366 LUA#61 26.5 23.5 39 93 765 795 829 876 725 785 785 741.5 NM_003581 LUA#62 12.5 28.5 72.5 52 69 62 68 55 56 51 62 58 NM_018115 LUA#63 32 44.5 66 163 1006 1121 1018 1181 1223 1257 1010 902 NM_021974 LUA#64 27.5 32 125 353 1802.5 1974.5 2019.5 2034 1663 1901.5 1782 1687 NM_024045 LUA#65 27.5 27.5 31 47 313.5 294 302 313 258 298 293.5 261 NM_004079 LUA#21 22.5 33 23 29 83 81.5 66 77 76 84 77 71 NM_000414 LUA#22 29 26 35 31 178 175 188 202 163 186 186 167 NM_001684 LUA#23 39 32 22 20.5 43 41 41.5 42 34 27 40 37 NM_003879 LUA#24 27 34 27.5 23 54 52.5 56 58 52 60 50 47 NM_002166 LUA#25 29 25 23 27 87 97 96 108 82 86 94 93 NM_005952 LUA#66 34 43.5 44.5 106 752 774 816 850 743.5 716.5 746 723 NM_001034 LUA#67 36 30 45 88 685 724 735 752 501 688 679 713 NM_003132 LUA#68 28 28 132 426 2030 2020 2077.5 2026.5 1779 1959 1928.5 1955 NM_018164 LUA#69 42.5 33 40 63 475 477 510 533 439 517 504 497 NM_014573 LUA#70 36 41.5 43 51 450 419 422 409 334 451 428 397 NM_014333 LUA#26 46 39 34 31 98 89 86 100 87 94 84 66 NM_006432 LUA#27 37 35 29 53.5 339 356 364 386 345 340 360 381 NM_000433 LUA#28 33 34 59 29 170 171 158 153 123 171 148 125 NM_000147 LUA#29 35.5 31 26 26 121 118 137 133 117 117 123 122.5 NM_000584 LUA#30 23 32 63 127 993 1017.5 1080.5 1142.5 950 993 1000 1062 NM_006452 LUA#71 49 36 33.5 34 140 135 121 128 105.5 135 117 112 NM_005915 LUA#72 34 30 35 29 114 124 126 128 116 135 122 110 NM_005980 LUA#73 39 32 76 270.5 1527 1547 1567 1651 1425 1597 1547 1462 NM_002539 LUA#74 43 35.5 117 366 2091 2193.5 2209 2175 1830 2082 2100 1912.5 NM_019058 LUA#75 35 31 62 157 1015 1152 1188 1217 952 1044 1044 1030 NM_004152 LUA#31 31 29.5 89.5 327 1999 1862 1854 1955 1630 2131 1980 1691 NM_004602 LUA#32 18 39.5 45 77.5 233.5 267 235 228.5 174 269.5 219 221 NM_018890 LUA#33 39 39 36 89 796.5 742 744 789.5 607 728.5 728 731.5 NM_001101 LUA#34 32.5 36.5 162.5 461 2101 2075 2065 2052.5 1748.5 2089.5 2074 1945 NM_006019 LUA#35 34 35 39 87 598 650 705 709 566 616 646 700 NM_004134 LUA#76 47.5 33 54 116 808 818 818 830 705 810 760.5 725 NM_005008 LUA#77 43 35 57 151 975.5 1026 1002.5 1038 842.5 1024 953 860 NM_020117 LUA#78 35 31.5 83 292 1653 1701 1746.5 1779 1445 1605.5 1611 1656 NM_001469 LUA#79 47 33.5 114 331 2049 2042 2027 2124 1772 2105.5 1958.5 1868 NM_021203 LUA#80 44 32 88 252 1671 1685 1722 1739 1458 1583.5 1673.5 1542 NM_002624 LUA#36 25 30 73 245.5 1176.5 1202.5 1226 1248 1132 1204 1139.5 1123 NM_004759 LUA#37 36 33 26 31 124 121 109 136 135 136 115 117 NM_002664 LUA#38 41 37 82.5 266 1521 1584 1621 1668 1378 1474 1502.5 1492 NM_000211 LUA#39 33 27 67 123.5 769.5 707 672 674 541 741.5 646 629.5 NM_002468 LUA#40 20 32.5 28 39 153 199 205 208.5 161 183 168 171.5 NM_000884 LUA#81 42 45 166 373 1693.5 1578 1629 1658 1421 1696 1631 1512 NM_003752 LUA#82 49 44 56 67 323 322 329 342 266 307.5 293.5 274 NM_018256 LUA#83 41 40 250 291 1045 1031 1078 1037.5 826 1007 961 985 NM_001948 LUA#84 40 40 35 42 213.5 203 219 225 180 203 201 195.5 NM_005566 LUA#85 39.5 44 199 520 2411.5 2445 2535.5 2462.5 2077 2326 2375 2334 NM_021103 LUA#41 30.5 38 97 247 1549 1351 1575 1693 1430.5 1500 1527.5 1296.5 NM_002970 LUA#42 36 45 24.5 52 522 484 507 532.5 440 542 529 519 NM_003332 LUA#43 35 35 60 178 1034.5 1140 1065 1157 988 1085 1058 1004 NM_004106 LUA#44 24 27 30.5 55 393 378 404 433.5 367 407 389 403 NM_002982 LUA#45 20 34 32 94 652 675 707 713.5 646 638 670 685 NM_005375 LUA#86 34.5 37 149.5 354 1811 1867 2001.5 1991.5 1718 1807 1813 1899 NM_000250 LUA#87 33 40 147.5 510 2353 2404 2415.5 2430 2078 2358 2296 2225 NM_004526 LUA#88 40 36 75 182.5 1064 1120 1093 1099 896 1035 1034 954.5 NM_004741 LUA#89 33 33 74 119.5 810 852 879.5 840 689.5 792 797.5 738 NM_002467 LUA#90 52.5 56 369 760 2507.5 2577 2640 2642 2322 2586 2604.5 2599 ACTB LUA#91 55.5 44 100 318 1796 1791 1930 1940.5 1558 1747.5 1799 1831 TFRC LUA#92 53 46.5 56 94 737 788 844 868.5 630 733 791 797 GAPDH_5 LUA#93 50 39 192 807 2708.5 2707 2729 2828 2320 2618 2741 2716 GAPDH_M LUA#94 43 42 201 737 3051 3052 3041 3075.5 2623 3060 2962 2834.5 GAPDH_3 LUA#95 45.5 41 616.5 1663 3524 3712 3728 3841 3284 3651.5 3806 3593 B Experiment 2- Tretinoin FlexMap description ID Tretinoin Tretinoin Tretinoin Tretinoin Tretinoin Tretinoin Tretinoin Tretinoin Tretinoin Tretinoin NM_005736 LUA#1 36.5 390 90.5 408 411 385 392 414.5 384.5 298.5 NM_000070 LUA#2 34 444 120 393 393 419 422 444 437.5 358 NM_018217 LUA#3 48 935 258 992 1053.5 947 966 1022.5 980 922 NM_004782 LUA#4 45 979.5 253 1005 1030 914 929 1044 1036.5 932 NM_014962 LUA#5 39 595.5 160 670.5 705 677.5 678 710 691 618 NM_004514 LUA#46 25 469 99 428 445 422 420.5 460 399 424.5 NM_006773 LUA#47 28 270 68 273 283 272 279 305 284 354.5 NM_014288 LUA#48 22 52 33 60 57 57 57 65 63.5 55 NM_017440 LUA#49 29 88 42 78 97 87.5 84 86.5 91 108 NM_007331 LUA#50 24 115 47 95.5 96 97.5 94.5 111.5 102 115 NM_173823 LUA#6 36 736 182.5 758 734.5 658.5 681 816 760 592 NM_000962 LUA#7 65 900 253 845 904.5 846 846 930 873 822.5 NM_003825 LUA#8 102 772 220 800 733 738 727 764 721 689.5 NM_016061 LUA#9 48 458 121 470.5 464 468 459 503.5 450 440 NM_000153 LUA#10 45 539 124 511 505 473 501 542 487 484 NM_006948 LUA#51 51 974 248 1014 1006 963.5 958.5 1024.5 980 887 NM_004631 LUA#52 38.5 391 97 396 399.5 399 401.5 424 399 397 NM_002358 LUA#53 33.5 103 39 109 103 96 97 119 109 94 NM_013402 LUA#54 30 82 46 90.5 95.5 85 84.5 97 89 83 NM_000875 LUA#55 28.5 81 36 79 80 85 90 87.5 89 104 NM_001974 LUA#11 30 516.5 92.5 533 515 493 470 539 494 340.5 NM_000632 LUA#12 26 491.5 96 406 400 360.5 374 395 369.5 476 NM_006457 LUA#13 43 115.5 65 115.5 118 120 131 117 131 121 NM_000698 LUA#14 64.5 1902 539 1934.5 1914.5 1773 1769 1871.5 1787 1685 NM_032571 LUA#15 22.5 244 58.5 228 234 208 205 239 228 244 NM_006138 LUA#56 32.5 115.5 48 111 127 118 119.5 120 124 117 NM_015201 LUA#57 27 266 63 252 243 230 244 268.5 241 245.5 NM_006985 LUA#58 33 55 33 50 52 54 60 63 56 50 NM_004095 LUA#59 31 287 77 286.5 293 270 294 331 293 227 NM_005914 LUA#60 42 871 213.5 1091 1131 1081.5 1125 1172.5 1161.5 1104 NM_007282 LUA#16 22 61 26 69 64 59 62 61 58 53 NM_003644 LUA#17 41 319 69 269 274 192 231 300 274 259 NM_001498 LUA#18 32 521 110.5 507.5 513 441.5 467 531.5 494.5 511 NM_003172 LUA#19 36 333 82 370 365 330 352 380 333 312.5 NM_004723 LUA#20 34 472.5 134.5 498 506 470 487.5 538 501 420 NM_014366 LUA#61 30 304.5 66 297 291 286.5 300 310.5 298.5 321 NM_003581 LUA#62 39 114 50 78 85 59.5 55 53 55 54 NM_018115 LUA#63 54 1440 356 1028 1210 935.5 1175 1004 1238 1270 NM_021974 LUA#64 49.5 1272 295 1368.5 1315 1193.5 1269 1365 1286.5 1160 NM_024045 LUA#65 29 163.5 48 164.5 175.5 158 157 182 178 149 NM_004079 LUA#21 28 144 49 133 133.5 142 143 152 150 147 NM_000414 LUA#22 34 547 115.5 596 561.5 550.5 543 598.5 564.5 544 NM_001684 LUA#23 30 98 38.5 66 79 68 77.5 83 71 75 NM_003879 LUA#24 22 93 39 91.5 86 84 82 97 95 96.5 NM_002166 LUA#25 30 237 60 230 240 221 218 242 227 254.5 NM_005952 LUA#66 40 265 65 274 260 263 224 268 243 261.5 NM_001034 LUA#67 32.5 280 65 281 253 271 252 276 260 246 NM_003132 LUA#68 37.5 721 157 690.5 680 633 648 716 635 618 NM_018164 LUA#69 34 205.5 61 182 188.5 182 186 211 198 200 NM_014573 LUA#70 32 187 49 179 161.5 172 157.5 198 178 154 NM_014333 LUA#26 41 706 166 712 720 662 633.5 726.5 720 704 NM_006432 LUA#27 52.5 1767 522 1718 1798 1725 1678 1814 1693 1842 NM_000433 LUA#28 34 810 158 860 842 753 724 823 786.5 574 NM_000147 LUA#29 36 1106 236 1132 1122.5 1051 1086 1171 1102 1135 NM_000584 LUA#30 72 2315 665 2389 2341.5 2247 2269 2450 2339.5 2517.5 NM_006452 LUA#71 27 83.5 39 93 90 88 96 90 86 86 NM_005915 LUA#72 30 47 33 47 43 44 49 54 47 47 NM_005980 LUA#73 33 197 52 212 204 187 188 209.5 202 190 NM_002539 LUA#74 33 437 89 423 409 392 368 441 416 397 NM_019058 LUA#75 35 192 55.5 181.5 179 176 171 194 177.5 194 NM_004152 LUA#31 74 2313 811 2471.5 2477.5 2269 2335.5 2470 2347 1882 NM_004602 LUA#32 42 320 116 337 334 363 355.5 296 303 274 NM_018890 LUA#33 38 1071 200 983 992 923.5 879 988 934 873 NM_001101 LUA#34 211 3046 1578 3029 3195 2934 2997 3139.5 3017 2733 NM_006019 LUA#35 36 1059 209.5 938.5 911.5 862 871 956 907 1033 NM_004134 LUA#76 36.5 423.5 90 417 415 415 383 426 406 372 NM_005008 LUA#77 34 452.5 108 501 482 432 439.5 502.5 452.5 393 NM_020117 LUA#78 35 750 149 739 734 606 654 748 697 734 NM_001469 LUA#79 73 1230 333.5 1437 1331.5 1308 1281.5 1360 1315 1107 NM_021203 LUA#80 37 757.5 152 703 701.5 650 660 741 657.5 657 NM_002624 LUA#36 71 1714 670 1664 1757 1580.5 1616 1772.5 1647.5 1643 NM_004759 LUA#37 26 284 60.5 179 207.5 192 195 201 206.5 235 NM_002664 LUA#38 91 2815 1002 2840.5 2805 2642 2652 2875 2701.5 2723 NM_000211 LUA#39 89 1828 470 1717 1734.5 1593.5 1570 1763 1639.5 1219 NM_002468 LUA#40 35 511 135 556.5 549 498 494 589.5 531.5 584 NM_000884 LUA#81 61 951 259.5 1004 988 925 916 1010 963.5 841 NM_003752 LUA#82 44 168 66 149.5 150 153 162 164 139 142 NM_018256 LUA#83 158 455 229 571 556 643 655 483.5 568 635.5 NM_001948 LUA#84 33 79.5 40 68 70.5 62 68 81 72 70 NM_005566 LUA#85 49 851 181.5 821 830 748 752 776.5 744 745 NM_021103 LUA#41 99 2331.5 939 2558 2559 793 1990 2996.5 2724 2581.5 NM_002970 LUA#42 42 1624 435.5 1759.5 1743 1655.5 1575 1823 1716.5 1563 NM_003332 LUA#43 120 2589.5 1244 2832 2821 2704 2692 2772 2733 2691.5 NM_004106 LUA#44 55 1762 508 1756 1799 1678 1587.5 1827 1697 1748 NM_002982 LUA#45 294 3328 2094 3522 3632 3562 3485 3768 3697 3859 NM_005375 LUA#86 78 552 158 568 593 585 589 587 565 642 NM_000250 LUA#87 39 249 70 246 243 232 239.5 253 236 228 NM_004526 LUA#88 31 244 69 270 260 240.5 243 277 256 233 NM_004741 LUA#89 57 370.5 99 329 324 317 325 328 327 402.5 NM_002467 LUA#90 108 762.5 205 792 798 823 776 759 741 823 ACTB LUA#91 107 2939 1020 2820 2870 2791 2727 2873.5 2807 2986 TFRC LUA#92 48 413 83 375 381 358 345.5 382 346 400.5 GAPDH_5 LUA#93 72 1965.5 509 1847 2001 1834.5 1691 1994 1888 1977.5 GAPDH_M LUA#94 73 1871 514 1911 2010.5 1693.5 1762.5 1932.5 1814 1595.5 GAPDH_3 LUA#95 139.5 2850 1137 3025 3066 2936 2973 3162.5 3075 2896

TABLE 8 A Experiment 3- Blank and DMSO description FlexMap ID BLANK BLANK DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO NM_005736 LUA#1 28 33.5 247 240.5 214.5 233 240 250 272 276 278.5 286.5 NM_000070 LUA#2 26 29.5 179 187.5 181 162.5 162 182.5 226 229 239.5 231 NM_018217 LUA#3 25 32 484 551.5 483 494.5 485 543 601 647.5 630 584 NM_004782 LUA#4 27.5 38.5 467 617 560.5 616 627.5 630 688 723.5 787.5 652.5 NM_014962 LUA#5 26.5 32 364.5 495 443 455 463 497 492 511 543 485.5 NM_004514 LUA#46 26 28 585 474.5 436 454 440 463 547 554.5 530 493 NM_006773 LUA#47 32 19 328 444 453 412 408 448.5 475.5 487 477 443 NM_014288 LUA#48 29 29 169 131.5 122.5 122 129 139.5 161 155.5 151.5 138 NM_017440 LUA#49 33.5 28 150 137 127.5 129 128 151 168 160.5 156 146 NM_007331 LUA#50 28 27.5 188 151 128 143 134.5 151 161 178 167 157 NM_173823 LUA#6 33.5 28 393 516 444 460.5 492 529 559.5 560 558 553.5 NM_000962 LUA#7 28 25 386.5 348 336 360 334 380 421.5 451 403.5 409 NM_003825 LUA#8 26 24.5 436 356 336.5 354.5 340 398 423 431 429.5 414 NM_016061 LUA#9 32 29 268 250 217 241.5 233.5 264 306 301 301 279 NM_000153 LUA#10 35 33 252 211.5 203 205 204 222 261 265 258 237 NM_006948 LUA#51 25 36 593 643 593 617 609 681 746 742 731.5 703.5 NM_004631 LUA#52 25 25 261 263 246.5 264 268.5 294 322 331.5 319 308 NM_002358 LUA#53 26 26 349 419 380 408 394.5 444 502 514 485 466 NM_013402 LUA#54 21 26.5 306 378 334.5 336.5 340 357 397.5 395 388 373 NM_000875 LUA#55 23.5 30 61 82 87 74 70 88 90 91 94 87 NM_001974 LUA#11 28 11 85 84 69 61 67 77 90 87 95.5 80 NM_000632 LUA#12 33 27 76 59 57 51 50 53 55.5 59 58 54 NM_006457 LUA#13 31.5 24 94 124 145 114 106 89 111 123 110 107 NM_000698 LUA#14 17 27.5 353 360.5 325 320 319 316.5 368 400 368 368 NM_032571 LUA#15 27 25 25.5 19 24 25.5 25 23 25 24 25 24 NM_006138 LUA#56 32 31 1076 1274 1213.5 1224 1176 1226 1316 1329 1312 1257 NM_015201 LUA#57 34 35 805.5 834 765.5 799 791 852.5 958 958 907 885.5 NM_006985 LUA#58 40 29.5 200 157 137 154 161 165 188 200 190 187 NM_004095 LUA#59 43 27 1904 1757.5 1707 1798 1644 1666 1925 2035 1825.5 1758.5 NM_005914 LUA#60 34 37 1376.5 1508 1561.5 1339 1246 1355 1448 1595 1420 1337 NM_007282 LUA#16 21 28 47 36 38 35.5 36 42 39 53 44 42 NM_003644 LUA#17 34 37 177.5 213 201 169.5 163 171 177 190 180.5 184 NM_001498 LUA#18 27 31 342 205 211 226 213.5 232 266 284 264 257 NM_003172 LUA#19 27 30 252 234 213 226 230.5 241 276 286.5 272.5 265.5 NM_004723 LUA#20 16.5 25.5 677 476 477.5 461 416 432.5 523 560.5 511 461 NM_014366 LUA#61 32.5 36.5 853 901 904 928 883 943.5 1045.5 1025.5 1014 934 NM_003581 LUA#62 26 24 280 66 48.5 39 41 50 42 46 50 40 NM_018115 LUA#63 31 39.5 1274 1143 1113 1353 1430 1382 1281 1275 1475.5 1295 NM_021974 LUA#64 38 40.5 1787 1842 1743 1855 1727 1723 2087 2161 1976 1987 NM_024045 LUA#65 28.5 30 255 265 267 262.5 251 268.5 303 311 296 298 NM_004079 LUA#21 36 33 73 69 64 60 67 74 87 68 99 71.5 NM_000414 LUA#22 25 42 240 180 157 170 162 184.5 198 198 200 184 NM_001684 LUA#23 25 24.5 26 29 28 27.5 37 36.5 35 38 35 39 NM_003879 LUA#24 21.5 18 56 44 50 50 49 47 54 59.5 54.5 54 NM_002166 LUA#25 28 29 75.5 91 97 103 89 94.5 108 107 104 98 NM_005952 LUA#66 38 27 561.5 627.5 648 673 600 640 672.5 738 687.5 650 NM_001034 LUA#67 26 30 575.5 674 631 619 595 641 764 768 739 692 NM_003132 LUA#68 27 21.5 1705 1840 1717 1797.5 1693.5 1803 2003 2030 1902.5 1861.5 NM_018164 LUA#69 37 27.5 511 495.5 484 532 507 569 633 645 655 594 NM_014573 LUA#70 36.5 40 463 485 425.5 418.5 431.5 478 534 552 501 510 NM_014333 LUA#26 33 28 89 79 94 81 75 89 86 89 83.5 79 NM_006432 LUA#27 26 26 302 287 273.5 302 292 317.5 349 360.5 335 338.5 NM_000433 LUA#28 23 36 187 137 111 115 117 133 153 150 136 136.5 NM_000147 LUA#29 32 34 110 120 117 129 126 125.5 142 148 140 137 NM_000584 LUA#30 29 29 1147 905 868 922 872.5 926 1019 1058 990 959.5 NM_006452 LUA#71 33 32 178 107 102 91 108 110.5 124 130 126 130.5 NM_005915 LUA#72 37 24 141.5 108 96 112 102 114 132 125 126.5 117 NM_005980 LUA#73 41 28 1314 1559 1544 1534 1467 1517 1660 1634.5 1617 1561 NM_002539 LUA#74 43 50 1863 1961.5 1903 2012.5 1865 1987 2241 2169.5 2041 2033 NM_019058 LUA#75 28.5 37.5 1168 1015 974 1004 959 957.5 1134 1130.5 1077 1035 NM_004152 LUA#31 34 32 1698 1990 1909 1973 1935.5 2211.5 2125 2252 2198 2153.5 NM_004602 LUA#32 25 22 206 222 198 216.5 213 229 275 261 279 255 NM_018890 LUA#33 30 44.5 703 648 591.5 627 607.5 669 715 737 695.5 690 NM_001101 LUA#34 22 23.5 2023.5 2026 1824 1841 1885 2013 2164 2148 2108 2048.5 NM_006019 LUA#35 38 34 489 556 511 540.5 528 535 631.5 620 610 583 NM_004134 LUA#76 26.5 26 953.5 677 576 622 589 620.5 715.5 744 688.5 681 NM_005008 LUA#77 33 37.5 882 839 752 791 785 832.5 942 961 951 884 NM_020117 LUA#78 38 43 1342 1519 1444.5 1498 1342 1459 1657 1641 1534 1457 NM_001469 LUA#79 40 43 1531 2065 1894.5 1953 1964.5 1969 2199 2216 2182 2115 NM_021203 LUA#80 39 45.5 1398 1482 1416 1418 1394 1424.5 1659 1692 1607 1533 NM_002624 LUA#36 27 24 1157.5 1111 1048.5 1073.5 1031 1095 1171.5 1194 1158 1135.5 NM_004759 LUA#37 34 24.5 115.5 84 84 87.5 102.5 140 130 108 150 114 NM_002664 LUA#38 35 29 1451 1230.5 1161 1253 1186 1241 1415 1470.5 1375 1311 NM_000211 LUA#39 34 35.5 778 580 496 516 551.5 624 688.5 724.5 690.5 671 NM_002468 LUA#40 27.5 20.5 145 144 156 164.5 168 161 168 206.5 177.5 181.5 NM_000884 LUA#81 39 43 1374 1662 1457.5 1477.5 1517.5 1579.5 1786 1770 1660 1608.5 NM_003752 LUA#82 40 44.5 206 265 245 260 232 223 257 273 246 241 NM_018256 LUA#83 35 32.5 583 948 927 859.5 840 833.5 885.5 923 915.5 934 NM_001948 LUA#84 30 31.5 171.5 166 151 152 142 158 172 175 169 167 NM_005566 LUA#85 39 23 2576 2426 2343.5 2313 2211.5 2208.5 2364 2414 2310 2268 NM_021103 LUA#41 29.5 34 1235.5 1639 1600 1483 1501 1430.5 1490 1618.5 1478 1415 NM_002970 LUA#42 25.5 28 353.5 489 460 492.5 480 537 579.5 614 565 566.5 NM_003332 LUA#43 35 38 954.5 987 937 1025 995 1066 1181 1206 1179 1122 NM_004106 LUA#44 21 26 373.5 394 372.5 394 375.5 419.5 457 461 420 418.5 NM_002982 LUA#45 32 31 603 673.5 609 665 596 652 735.5 760 709.5 655 NM_005375 LUA#86 39.5 33 1128 1776 1693 1718.5 1608 1662 1785.5 1801 1732.5 1749 NM_000250 LUA#87 45 38 2308 1891 1773 1821 1758 1852 2090 2148.5 2015 1937 NM_004526 LUA#88 42 30 1019 1079 955 1021 955 1007 1114 1164 1092 1040 NM_004741 LUA#89 33 43.5 623 778 763 713 731 813 816 821 808 773 NM_002467 LUA#90 40 44 1658 2414 2353 2281 2242.5 2287.5 2464 2519.5 2469.5 2358 ACTB LUA#91 37 42.5 1668 1753 1743 1832 1683 1785 1924 1902 1857 1793 TFRC LUA#92 59.5 51 543 595 578 659 620 658.5 750 715 718 678 GAPDH_5 LUA#93 42 51 1954 3132.5 2965 2946 2848 2897 2953 2930 2799 2733.5 GAPDH_M LUA#94 41 45.5 2721 3317 3109 3039 2963 3139 3320 3320 3195 3068.5 GAPDH_3 LUA#95 47.5 45 2788.5 3887 3821 3905 3912.5 3908.5 4244.5 4050.5 4090 4030 B Experiment 3- Tretinoin description FlexMap ID Tretinoin Tretinoin Tretinoin Tretinoin Tretinoin Tretinoin Tretinoin Tretinoin Tretinoin Tretinoin NM_005736 LUA#1 55 84 113 205.5 298 336 235 38 236.5 280 NM_000070 LUA#2 54 82.5 118 224 328.5 330 254 42 274 303 NM_018217 LUA#3 109 187.5 275.5 571 779 801 582.5 61 690.5 742 NM_004782 LUA#4 105 184.5 279 539 825.5 866 689 62 764 803.5 NM_014962 LUA#5 82 120 188.5 369 544.5 551.5 435 52.5 474.5 507 NM_004514 LUA#46 47 73 120 233.5 284.5 300.5 206 33 287 281 NM_006773 LUA#47 37 51 74.5 133 213 245 189.5 34 243 218 NM_014288 LUA#48 30 25 30.5 39 48 48 43 33 45 48 NM_017440 LUA#49 34 34.5 41 74 72 98 77 31 102 86.5 NM_007331 LUA#50 29 40.5 43 65 87 88 81 28 81.5 79 NM_173823 LUA#6 93 147.5 213 415 597 627.5 479 51.5 517 573 NM_000962 LUA#7 142 230 296 559 717.5 722.5 545.5 98.5 665 712 NM_003825 LUA#8 172 244 276 446 630 617 500 162.5 575 613 NM_016061 LUA#9 78 117 154 268 406 421 311 60 359 392 NM_000153 LUA#10 69.5 108 141.5 277 400 396 292 61 336 378 NM_006948 LUA#51 111 189 284 558.5 772 781 604 63 709.5 736 NM_004631 LUA#52 56 78 109 213.5 315.5 325 269 45 306 303 NM_002358 LUA#53 31.5 39 42 68 100 100 76 33 87 93 NM_013402 LUA#54 29 29.5 44.5 56.5 73 76 63 30 68 75 NM_000875 LUA#55 27.5 33 41 59 60 70 70 26 75 70 NM_001974 LUA#11 41 68 94 202 344 371 253 32 276 318.5 NM_000632 LUA#12 40 56 89 189 272 293 236 33 277.5 300 NM_006457 LUA#13 50 53.5 63 82 102 111.5 91 42 89 95 NM_000698 LUA#14 188 330 526 1026 1241 1325 997 65 1148 1215.5 NM_032571 LUA#15 29 39 56 98 152 157 121 30.5 132 146 NM_006138 LUA#56 37 40 49 67 97 102.5 86 38 87 95 NM_015201 LUA#57 40 49 64 139 197 228 156 31 208 198 NM_006985 LUA#58 31 32 37.5 45.5 52 59 46.5 30 48 59 NM_004095 LUA#59 46 66 86.5 166.5 221 208.5 147 36 169 205 NM_005914 LUA#60 102 218.5 314 594 914 889 716.5 44 525.5 804.5 NM_007282 LUA#16 27 29 30 42 53 61.5 58 28 46 62.5 NM_003644 LUA#17 53 72 75.5 137 220 238.5 179 39 181.5 218 NM_001498 LUA#18 40 71 121 262 386 382 279 30 397 391.5 NM_003172 LUA#19 45 68 97 199 234 250 179 33 208 227 NM_004723 LUA#20 61 105 151 271.5 350 352 258 37 281 329 NM_014366 LUA#61 38 61 89.5 191 291.5 299.5 235 33.5 217 294 NM_003581 LUA#62 40 36 42 47 46.5 46 40 33 40.5 39.5 NM_018115 LUA#63 109.5 234 409 710.5 896 1206.5 927 61 1217 860.5 NM_021974 LUA#64 136.5 255.5 403 781.5 929 940 667.5 55 776 891 NM_024045 LUA#65 35 40 57 92.5 135.5 137 110 29 112 127.5 NM_004079 LUA#21 34 41 53 88 122 124 121 30 113 124 NM_000414 LUA#22 48 74 114 272.5 479 477.5 378 35 384 448 NM_001684 LUA#23 30 31.5 31 52 66 60 49 25 52.5 60 NM_003879 LUA#24 23 29 36 51 77 76 59 34 65 69 NM_002166 LUA#25 37 51 74 132 188.5 211 170 29 169.5 207 NM_005952 LUA#66 43 56 74 143 200 197.5 156 34 204 199 NM_001034 LUA#67 42 47 73 122 178 184 155 33 170 171 NM_003132 LUA#68 74 117.5 194 367 461 482 344 38 431 428 NM_018164 LUA#69 37 46 67 139 150 156.5 133 36 148.5 149 NM_014573 LUA#70 40 45 56 93 156 161.5 123 38.5 138.5 144.5 NM_014333 LUA#26 74 128 211 427 674 705 572 41 613 656.5 NM_006432 LUA#27 190 358.5 574 1118 1397.5 1403 1160 68 1364 1394 NM_000433 LUA#28 62 105 169 371.5 580.5 600.5 406 32 454 548 NM_000147 LUA#29 95 188.5 323 713 1109 1156 905 43 987 1072 NM_000584 LUA#30 240 426 663 1369.5 1886 1908.5 1555 96.5 1878.5 1840 NM_006452 LUA#71 25 37 37.5 43 57 71 44 23.5 52 60 NM_005915 LUA#72 25 27 30 35 40 37 38 29 46 42 NM_005980 LUA#73 32 43 52 102.5 157 166 132 34 160 157 NM_002539 LUA#74 46 74 106 209 271 323.5 225 36 276 278.5 NM_019058 LUA#75 37 48 56 90 126 137 97 34 102 129 NM_004152 LUA#31 319 601 837 1645 2020 2157.5 1685 90.5 1845 1993 NM_004602 LUA#32 61 87.5 116 185 270 293 288 46 255 274.5 NM_018890 LUA#33 82 151.5 240 534.5 760 762 652 41 737 769 NM_001101 LUA#34 799 1279 1711 2759.5 2895 2880 2335 235 2622 2644 NM_006019 LUA#35 73 140 227 497 747 787.5 655 34 810 792 NM_004134 LUA#76 47.5 67 93 182 284 297 240 36 268 286 NM_005008 LUA#77 53.5 87 114 239.5 315 316 232 41 242 301.5 NM_020117 LUA#78 65 114 184 371 519 543 415 45 487 502 NM_001469 LUA#79 141.5 246.5 395 731.5 1124 1134 921 75.5 988.5 1104 NM_021203 LUA#80 65 109 165 355 480 514 369 46 445.5 471 NM_002624 LUA#36 253.5 500 700.5 1234 1402 1395 1167.5 79 1359 1343 NM_004759 LUA#37 32 41 60 119 132.5 213 159 28 242 139.5 NM_002664 LUA#38 383 702 1051.5 1832 2052 2064 1635.5 101 1922 1934 NM_000211 LUA#39 204.5 356 501 939 1215 1215 924.5 109 1072 1160 NM_002468 LUA#40 57.5 93 132.5 303 450 473.5 357 36.5 367 421 NM_000884 LUA#81 126 209 304 612 794.5 804 624 58 670 692 NM_003752 LUA#82 54 54 66 93 105 123 96 41 116 113 NM_018256 LUA#83 178.5 177 268 371 633 804.5 779 151 638.5 688.5 NM_001948 LUA#84 34 38 38 50 61 59 54 33 63 62 NM_005566 LUA#85 89 117 183 197.5 597 632 489 45 539.5 610.5 NM_021103 LUA#41 467.5 781.5 992 1953 2465.5 2408 495 142 629 2096 NM_002970 LUA#42 164 329 528 1106 1508 1570.5 1247 57 1316 1443 NM_003332 LUA#43 591 1003 1446 2239 2492 2467 2041 177.5 2312 2346.5 NM_004106 LUA#44 235 457 672 1276 1500 1506 1234 68 1458 1423 NM_002982 LUA#45 1175 1759 2328 3359 3548 3612.5 3101 373 3449 3440 NM_005375 LUA#86 106 131 196 334 547.5 602 531 86 548 553 NM_000250 LUA#87 46 52 67 115 159 166 131 38 141 157 NM_004526 LUA#88 43 61 76.5 137 187.5 191 138 37 153.5 172 NM_004741 LUA#89 70 77 131 204.5 315 415.5 300.5 58 397 326 NM_002467 LUA#90 136 162 239 409.5 576 644 527 86 571 552.5 ACTB LUA#91 452 812 1191 2112.5 2760 2845 2391 144.5 2538 2604.5 TFRC LUA#92 54 66 90 168 256 272 213 45 252 255 GAPDH_5 LUA#93 261.5 439.5 741 1388 1787 1865 1714 97 1699 1739.5 GAPDH_M LUA#94 221.5 396 590.5 1179.5 1602 1586 1267.5 79 1342.5 1462 GAPDH_3 LUA#95 579 1017 1438 2495.5 2680 2718 2148 172 2330.5 2534

TABLE 9 Sample Information Field Description Name Sample name used in this study Data Set Data set that stores the miRNA expression data; 1 for miGCM, 2 for PDT_miRNA, 3 for mLung, 4 for ALL, 5 for HL60, 6 for erythroid SR Name Corresponding sample name in Ramaswamy et al, PNAS, 2001, 98: 15149-15154; empty entry for no match HuFL Scan Scan name for Affymetrix HuFL (Hu6800) chip, if available Hu35KsubA Scan name for Affymetrix Hu35KsubA chip, if available Scan BV Bead version that is used to detect the sample SSC Sample source code; 1 for Ramaswamy study, 2 for St Jude, 3 for Dana-Farber, 4 for MIT MAL Maliganancy status code; 1 for Normal, 2 for Tumor, 3 for cell line TT Tissue type code; 1 for stomach, 2 for colon, 3 for pancreas, 4 for liver, 5 for kidney, 6 for bladder, 7 for prostate, 8 for ovary, 9 for uterus, 10 for human lung, 11 for mesothelioma, 12 for melanoma, 13 for breast, 14 for brain, 19 for B cell ALL, 20 for T cell ALL, 21 for follicular cleaved lymphoma, 22 for large B cell lymphoma, 23 for mycosis fungoidis, 24 for acute myelogenous leukemia, 26 for mouse lung, 27 for erythrocytes CLT Cell line type code; 1 for non-cell-line/others, 2 for MCF-7, 3 for SKMEL-5, 4 for PC-3, 5 for K562, 6 for HEL, 7 for TF-1, 8 for 293, 9 for HL60, 10 for T-ALL cell lines PDT Poorly differentiated tumor (PDT) code; 0 for others, 1 for PDT used in prediction, 2 for PDT not used in prediction due to lack of successful Affymetrix scans AS ALL Subtype; 0 for others or unknowns, 1 for BCR/ABL, 2 for E2A/PBX1, 3 for Hyperdiploid 47 to 50, 4 for Hyperdiploid >50, 5 for MLL, 6 for T_ALL, 7 for TEL/AML1, 9 for Normal ploidy EP Epithelial code; 0 for others, 1 for epithelial sample GI Gastrointestinal tract code; 0 for others and cell lines, 1 for GI sample Culture Description of culture condition for HL-60 and erythrocyte differentiation experiments N-T CLS Sample used to build the normal/tumor classifier; 0 for others, 1 for used MultiC CLS Sample used to build the multi-cancer classifier; 0 for others, 1 for used RNA Starting quantity of total RNA for profiling, measured in micrograms Data Hu35KsubA N-T MultiC Name Set SR Name HuFL Scan Scan BV SSC MAL TT CLT PDT AS EP GI Culture CLS CLS RNA N_STOM_1 1 1 1 1 1 1 0 0 1 1 NA 0 0 10 N_STOM_2 1 1 1 1 1 1 0 0 1 1 NA 0 0 10 N_STOM_3 1 1 1 1 1 1 0 0 1 1 NA 0 0 10 N_STOM_4 1 1 1 1 1 1 0 0 1 1 NA 0 0 10 N_STOM_5 1 1 1 1 1 1 0 0 1 1 NA 0 0 10 N_STOM_6 1 1 1 1 1 1 0 0 1 1 NA 0 0 10 N_COLON_1 1 CL2000090529AA CL2000090729AA 1 1 1 2 1 0 0 1 1 NA 1 0 10 N_COLON_2 1 1 1 1 2 1 0 0 1 1 NA 1 0 10 N_COLON_3 1 CL2000091210AA CL2000091510AA 1 1 1 2 1 0 0 1 1 NA 1 0 10 N_COLON_4 1 CL2000090527AA CL2000090727AA 1 1 1 2 1 0 0 1 1 NA 1 0 10 N_COLON_5 1 CL2000090523AA CL2000090723AA 1 1 1 2 1 0 0 1 1 NA 1 0 10 T_COLON_1 1 1 1 2 2 1 0 0 1 1 NA 1 0 10 T_COLON_2 1 Colorectal_Adeno_mCRT2_(9752) CH2000030408AA SR2000042821AA 1 1 2 2 1 0 0 1 1 NA 1 1 10 T_COLON_3 1 Colorectal_Adeno_9912c055_CC CH2000031308AA SR2000042828AA 1 1 2 2 1 0 0 1 1 NA 1 1 10 T_COLON_4 1 Colorectal_Adeno_95_I_175 CH2000030516AA SR2000042819AA 1 1 2 2 1 0 0 1 1 NA 1 1 10 T_COLON_5 1 Colorectal_Adeno_0001c038_CC CH2000031317AA SR2000042826AA 1 1 2 2 1 0 0 1 1 NA 1 1 10 T_COLON_6 1 1 1 2 2 1 0 0 1 1 NA 1 0 10 T_COLON_7 1 Colorectal_Adeno_95_I_057 CH2000030507AA SR2000042824AA 1 1 2 2 1 0 0 1 1 NA 1 1 10 T_COLON_8 1 SR2000051017AA 1 1 2 2 1 0 0 1 1 NA 1 0 10 T_COLON_9 1 Colorectal_Adeno_0001c040_CC CH2000031309AA CL2000091537AA 1 1 2 2 1 0 0 1 1 NA 1 1 10 T_COLON_10 1 Colorectal_Adeno_HCTN_CRT1_(18851_A1B) SR1999121605AA SR2000042825AA 1 1 2 2 1 0 0 1 1 NA 1 1 10 N_PAN_1 1 CL2000090543AA CL2000090743AA 1 1 1 3 1 0 0 1 1 NA 0 0 10 T_PAN_1 1 Pancreas_Adeno_Pan_3T CH2000031008AA SR2000042222AA 1 1 2 3 1 0 0 1 1 NA 0 1 10 T_PAN_2 1 Pancreas_Adeno_Pan_6T CH2000031312AA SR2000042224AA 1 1 2 3 1 0 0 1 1 NA 0 1 10 T_PAN_3 1 Pancreas_Adeno_97_I_077 CH2000031020AA 1 1 2 3 1 0 0 1 1 NA 0 0 10 T_PAN_4 1 Pancreas_Adeno_Pan_2T CH2000031318AA SR2000042221AA 1 1 2 3 1 0 0 1 1 NA 0 1 10 T_PAN_5 1 Pancreas_Adeno_Pan_7T CH2000031311AA SR2000042225AA 1 1 2 3 1 0 0 1 1 NA 0 1 10 T_PAN_6 1 Pancreas_Adeno_Pan_17T CL2000071414AA CL2000071840AA 1 1 2 3 1 0 0 1 1 NA 0 1 10 T_PAN_7 1 Pancreas_Adeno_Pan_4T CH2000031024AA SR2000042223AA 1 1 2 3 1 0 0 1 1 NA 0 1 10 T_PAN_8 1 Pancreas_Adeno_Pan_1T CH2000031306AA SR2000042220AA 1 1 2 3 1 0 0 1 1 NA 0 1 10 T_PAN_9 1 Pancreas_Adeno_Pan_29T CL2000071409AA CL2000081524AA 1 1 2 3 1 0 0 1 1 NA 0 1 10 N_LVR_1 1 1 1 1 4 1 0 0 1 1 NA 0 0 10 N_LVR_2 1 1 1 1 4 1 0 0 1 1 NA 0 0 10 N_LVR_3 1 1 1 1 4 1 0 0 1 1 NA 0 0 10 N_KID_1 1 CL2000091226AA CL2000091526AA 1 1 1 5 1 0 0 1 0 NA 1 0 10 N_KID_2 1 CL2000090539AA CL2000090739AA 1 1 1 5 1 0 0 1 0 NA 1 0 10 N_KID_3 1 CL2000091214AA CL2000091514AA 1 1 1 5 1 0 0 1 0 NA 1 0 10 T_KID_1 1 Renal_Carcinoma_Carc_628TG_(—) MG1999030902AA SR2000060917AA 1 1 2 5 1 0 0 1 0 NA 1 1 10 T_KID_2 1 SR2000060913AA 1 1 2 5 1 0 0 1 0 NA 1 0 10 T_KID_3 1 Renal_Carcinoma_Carc_614TO_(—) MG1999030904AA SR2000060914AA 1 1 2 5 1 0 0 1 0 NA 1 1 10 T_KID_4 1 Renal_Carcinoma_Carc_609TO_(—) MG1999030901AA SR2000060916AA 1 1 2 5 1 0 0 1 0 NA 1 1 10 T_KID_5 1 Renal_Carcinoma_92_I_126 CH2000030508AA SR2000050421AA 1 1 2 5 1 0 0 1 0 NA 1 1 10 TCL_293_1 1 1 4 3 5 8 0 0 1 0 NA 0 0 10 TCL_293_2 1 1 4 3 5 8 0 0 1 0 NA 0 0 10 TCL_293_3 1 1 4 3 5 8 0 0 1 0 NA 0 0 10 N_BLDR_1 1 CL2000090532AA CL2000090732AA 1 1 1 6 1 0 0 1 0 NA 0 0 10 N_BLDR_2 1 1 1 1 6 1 0 0 1 0 NA 0 0 10 T_BLDR_1 1 Bladder_TCC_9858 SR2000042208AA SR2000051014AA 1 1 2 6 1 0 0 1 0 NA 0 1 10 T_BLDR_2 1 1 1 2 6 1 0 0 1 0 NA 0 0 10 T_BLDR_3 1 Bladder_TCC_11520 SR2000042201AA SR2000051005AA 1 1 2 6 1 0 0 1 0 NA 0 1 10 T_BLDR_4 1 Bladder_TCC_B_0004 CL2000080113AA CL2000080314AA 1 1 2 6 1 0 0 1 0 NA 0 1 10 T_BLDR_5 1 Bladder_TCC_B_0008 CL2000080115AA CL2000080803AA 1 1 2 6 1 0 0 1 0 NA 0 1 10 T_BLDR_6 1 Bladder_TCC_B_0001 CL2000080110AA CL2000080311AA 1 1 2 6 1 0 0 1 0 NA 0 1 10 T_BLDR_7 1 Bladder_TCC_07-B_003E CL2000080109AA CL2000080310AA 1 1 2 6 1 0 0 1 0 NA 0 1 10 N_PROST_1 1 CL2000090515AA CL2000090715AA 1 1 1 7 1 0 0 1 0 NA 1 0 10 N_PROST_2 1 CL2000090518AA CL2000090718AA 1 1 1 7 1 0 0 1 0 NA 1 0 10 N_PROST_3 1 1 1 1 7 1 0 0 1 0 NA 1 0 10 N_PROST_4 1 CL2000090514AA CL2000090714AA 1 1 1 7 1 0 0 1 0 NA 1 0 10 N_PROST_5 1 1 1 1 7 1 0 0 1 0 NA 1 0 10 N_PROST_6 1 CL2000090517AA CL2000090717AA 1 1 1 7 1 0 0 1 0 NA 1 0 10 N_PROST_7 1 CL2000090519AA CL2000090719AA 1 1 1 7 1 0 0 1 0 NA 1 0 10 N_PROST_8 1 CL2000090516AA CL2000090716AA 1 1 1 7 1 0 0 1 0 NA 1 0 10 T_PROST_1 1 Prostate_Adeno_P_0025 CL2000090506AA CL2000090706AA 1 1 2 7 1 0 0 1 0 NA 1 1 10 T_PROST_2 1 Prostate_Adeno_P_0030 CL2000090507AA CL2000090707AA 1 1 2 7 1 0 0 1 0 NA 1 1 10 T_PROST_3 1 Prostate_Adeno_P_0036 CL2000090509AA CL2000090709AA 1 1 2 7 1 0 0 1 0 NA 1 1 10 T_PROST_4 1 Prostate_Adeno_P_0033 CL2000090508AA CL2000090708AA 1 1 2 7 1 0 0 1 0 NA 1 1 10 T_PROST_5 1 Prostate_Adeno_95_I_256 CL2000071413AA CL2000071839AA 1 1 2 7 1 0 0 1 0 NA 1 1 10 T_PROST_6 1 Prostate_Adeno_94_I_052 CH2000030405AA SR2000050409AA 1 1 2 7 1 0 0 1 0 NA 1 1 10 TCL_PC- 1 1 4 3 7 4 0 0 1 0 NA 0 0 10 3_1 TCL_PC- 1 1 4 3 7 4 0 0 1 0 NA 0 0 10 3_2 TCL_PC- 1 1 4 3 7 4 0 0 1 0 NA 0 0 10 3_3 TCL_PC- 1 1 4 3 7 4 0 0 1 0 NA 0 0 10 3_4 T_OVARY_1 1 Ovary_Adeno_mOVT1_(8691) CH2000030411AA SR2000050412AA 1 1 2 8 1 0 0 1 0 NA 0 1 10 T_OVARY_2 1 1 1 2 8 1 0 0 1 0 NA 0 0 10 T_OVARY_3 1 Ovary_Adeno_H_6206 CL2000080107AA CL2000080308AA 1 1 2 8 1 0 0 1 0 NA 0 1 10 T_OVARY_4 1 Ovary_Adeno_07-B_001B CL2000080103AA CL2000080304AA 1 1 2 8 1 0 0 1 0 NA 0 1 10 T_OVARY_5 1 Ovary_Adeno_07-B_014G CL2000080104AA CL2000080305AA 1 1 2 8 1 0 0 1 0 NA 0 1 10 T_OVARY_6 1 Ovary_Adeno_93_I_081 CH2000030415AA SR2000050411AA 1 1 2 8 1 0 0 1 0 NA 0 1 10 T_OVARY_7 1 1 1 2 8 1 0 0 1 0 NA 0 0 10 N_UT_1 1 1 1 1 9 1 0 0 1 0 NA 1 0 10 N_UT_2 1 1 1 1 9 1 0 0 1 0 NA 1 0 10 N_UT_3 1 1 1 1 9 1 0 0 1 0 NA 1 0 10 N_UT_4 1 1 1 1 9 1 0 0 1 0 NA 1 0 10 N_UT_5 1 1 1 1 9 1 0 0 1 0 NA 1 0 10 N_UT_6 1 1 1 1 9 1 0 0 1 0 NA 1 0 10 N_UT_7 1 1 1 1 9 1 0 0 1 0 NA 1 0 10 N_UT_8 1 CL2000091225AA CL2000091525AA 1 1 1 9 1 0 0 1 0 NA 1 0 10 N_UT_9 1 1 1 1 9 1 0 0 1 0 NA 1 0 10 T_UT_1 1 Uterus_Adeno_2967 SR2000042205AA SR2000051008AA 1 1 2 9 1 0 0 1 0 NA 1 1 10 T_UT_2 1 Uterus_Adeno_3663 SR2000042203AA SR2000051003AA 1 1 2 9 1 0 0 1 0 NA 1 1 10 T_UT_3 1 Uterus_Adeno_3226 SR2000042207AA SR2000051931AA 1 1 2 9 1 0 0 1 0 NA 1 1 10 T_UT_4 1 Uterus_Adeno_4915 SR2000042209AA SR2000051001AA 1 1 2 9 1 0 0 1 0 NA 1 1 10 T_UT_5 1 Uterus_Adeno_92_I_073 CH2000030413AA SR2000050424AA 1 1 2 9 1 0 0 1 0 NA 1 1 10 T_UT_6 1 Uterus_Adeno_5116 SR2000042206AA SR2000051016AA 1 1 2 9 1 0 0 1 0 NA 1 1 10 T_UT_7 1 Uterus_Adeno_4075 SR2000042212AA SR2000051010AA 1 1 2 9 1 0 0 1 0 NA 1 1 10 T_UT_8 1 Uterus_Adeno_2552 SR2000042210AA SR2000051004AA 1 1 2 9 1 0 0 1 0 NA 1 1 10 T_UT_9 1 Uterus_Adeno_4203 SR2000042202AA SR2000051009AA 1 1 2 9 1 0 0 1 0 NA 1 1 10 T_UT_10 1 Uterus_Adeno_4840 SR2000042214AA SR2000051011AA 1 1 2 9 1 0 0 1 0 NA 1 1 10 N_LUNG_1 1 CL2000090521AA CL2000090721AA 1 1 1 10 1 0 0 1 0 NA 1 0 10 N_LUNG_2 1 1 1 1 10 1 0 0 1 0 NA 1 0 10 N_LUNG_3 1 CL2000091223AA CL2000091523AA 1 1 1 10 1 0 0 1 0 NA 1 0 10 N_LUNG_4 1 1 1 1 10 1 0 0 1 0 NA 1 0 10 T_LUNG_1 1 Lung_Adeno_004_B CL2000090501AA CL2000090701AA 1 1 2 10 1 0 0 1 0 NA 1 1 10 T_LUNG_2 1 Lung_Adeno_H_20154 CL2000090504AA CL2000090704AA 1 1 2 10 1 0 0 1 0 NA 1 1 10 T_LUNG_3 1 Met_Lung_H_20300 CL2000090505AA CL2000090705AA 1 1 2 10 1 0 0 1 0 NA 1 1 10 T_LUNG_4 1 Lung_Adeno_009_C CL2000090502AA CL2000090702AA 1 1 2 10 1 0 0 1 0 NA 1 1 10 T_LUNG_5 1 1 1 2 10 1 0 0 1 0 NA 1 0 10 T_LUNG_6 1 Lung_Adeno_H_20387 CL2000090503AA CL2000090703AA 1 1 2 10 1 0 0 1 0 NA 1 1 10 T_MESO_1 1 Mesothelioma_300_T CH2000031101AA SR2000050516AA 1 1 2 11 1 0 0 1 0 NA 0 1 10 T_MESO_2 1 Mesothelioma_224_T5 CH2000031015AA SR2000050509AA 1 1 2 11 1 0 0 1 0 NA 0 1 10 T_MESO_3 1 Mesothelioma_235_T6 CH2000031018AA SR2000050507AA 1 1 2 11 1 0 0 1 0 NA 0 1 10 T_MESO_4 1 Mesothelioma_169_T7 CH2000031004AA SR2000050501AA 1 1 2 11 1 0 0 1 0 NA 0 1 10 T_MESO_5 1 Mesothelioma_31_T10 CH2000031014AA SR2000050513AA 1 1 2 11 1 0 0 1 0 NA 0 1 10 T_MESO_6 1 Mesothelioma_165_T5 CH2000031019AA SR2000050510AA 1 1 2 11 1 0 0 1 0 NA 0 1 10 T_MESO_7 1 Mesothelioma_74_T6 CH2000031021AA SR2000050514AA 1 1 2 11 1 0 0 1 0 NA 0 1 10 T_MESO_8 1 Mesothelioma_215_T5 CH2000031017AA SR2000050511AA 1 1 2 11 1 0 0 1 0 NA 0 1 10 T_MELA_1 1 Melanoma_96_I_166 CH2000031316AA SR2000050518AA 1 1 2 12 1 0 0 1 0 NA 0 1 10 T_MELA_2 1 Melanoma_94_I_149 CH2000031011AA SR2000050504AA 1 1 2 12 1 0 0 1 0 NA 0 1 10 T_MELA_3 1 Melanoma_93_I_262 CH2000031305AA SR2000050519AA 1 1 2 12 1 0 0 1 0 NA 0 1 10 TCL_SKMEL- 1 1 4 3 12 3 0 0 1 0 NA 0 0 10 5_1 TCL_SKMEL- 1 1 4 3 12 3 0 0 1 0 NA 0 0 10 5_2 N_BRST_1 1 CL2000090513AA CL2000090713AA 1 1 1 13 1 0 0 1 0 NA 1 0 10 N_BRST_2 1 CL2000090511AA CL2000090711AA 1 1 1 13 1 0 0 1 0 NA 1 0 10 N_BRST_3 1 CL2000090512AA CL2000090712AA 1 1 1 13 1 0 0 1 0 NA 1 0 10 T_BRST_1 1 Breast_Adeno_9912c068_CC CH2000031302AA SR2000042806AA 1 1 2 13 1 0 0 1 0 NA 1 1 10 T_BRST_2 1 Breast_Adeno_94_I_155 CH2000030407AA SR2000042804AA 1 1 2 13 1 0 0 1 0 NA 1 1 10 T_BRST_3 1 Breast_Adeno_mBRT1_(8697) CH2000030509AA SR2000051018AA 1 1 2 13 1 0 0 1 0 NA 1 1 10 T_BRST_4 1 Breast_Adeno_95_I_029 CH2000030511AA SR2000042803AA 1 1 2 13 1 0 0 1 0 NA 1 1 10 T_BRST_5 1 Breast_Adeno_93_I_250 CH2000031102AA SR2000042807AA 1 1 2 13 1 0 0 1 0 NA 1 1 10 T_BRST_6 1 Breast_Adeno_09-B_003A CL2000080301AA CL2000091505AA 1 1 2 13 1 0 0 1 0 NA 1 1 10 TCL_MCF- 1 1 4 3 13 2 0 0 1 0 NA 0 0 10 7_1 TCL_MCF- 1 1 4 3 13 2 0 0 1 0 NA 0 0 10 7_2 TCL_MCF- 1 1 4 3 13 2 0 0 1 0 NA 0 0 10 7_3 TCL_MCF- 1 1 4 3 13 2 0 0 1 0 NA 0 0 10 7_4 TCL_MCF- 1 1 4 3 13 2 0 0 1 0 NA 0 0 10 7_5 N_BRAIN_1 1 CL2000091228AA CL2000091528AA 1 1 1 14 1 0 0 0 0 NA 0 0 10 N_BRAIN_2 1 CL2000090547AA CL2000090747AA 1 1 1 14 1 0 0 0 0 NA 0 0 10 T_BALL_1 1 1 2 2 19 1 0 0 0 0 NA 0 0 5 T_BALL_2 1 1 2 2 19 1 0 0 0 0 NA 0 0 5 T_BALL_3 1 1 2 2 19 1 0 0 0 0 NA 0 0 5 T_BALL_4 1 1 2 2 19 1 0 0 0 0 NA 0 0 5 T_BALL_5 1 1 2 2 19 1 0 0 0 0 NA 0 0 5 T_BALL_6 1 1 2 2 19 1 0 0 0 0 NA 0 0 5 T_BALL_7 1 1 2 2 19 1 0 0 0 0 NA 0 0 5 T_BALL_8 1 1 2 2 19 1 0 0 0 0 NA 0 0 5 T_BALL_9 1 1 2 2 19 1 0 0 0 0 NA 0 0 5 T_BALL_10 1 1 2 2 19 1 0 0 0 0 NA 0 0 5 T_BALL_11 1 1 2 2 19 1 0 0 0 0 NA 0 0 5 T_BALL_12 1 1 2 2 19 1 0 0 0 0 NA 0 0 5 T_BALL_13 1 1 2 2 19 1 0 0 0 0 NA 0 0 5 T_BALL_14 1 1 2 2 19 1 0 0 0 0 NA 0 0 5 T_BALL_15 1 1 2 2 19 1 0 0 0 0 NA 0 0 5 T_BALL_16 1 1 2 2 19 1 0 0 0 0 NA 0 0 5 T_BALL_17 1 1 2 2 19 1 0 0 0 0 NA 0 0 5 T_BALL_18 1 1 2 2 19 1 0 0 0 0 NA 0 0 5 T_BALL_19 1 1 2 2 19 1 0 0 0 0 NA 0 0 5 T_BALL_20 1 1 2 2 19 1 0 0 0 0 NA 0 0 5 T_BALL_21 1 1 2 2 19 1 0 0 0 0 NA 0 0 5 T_BALL_22 1 1 2 2 19 1 0 0 0 0 NA 0 0 5 T_BALL_23 1 1 2 2 19 1 0 0 0 0 NA 0 0 5 T_BALL_24 1 1 2 2 19 1 0 0 0 0 NA 0 0 5 T_BALL_25 1 1 2 2 19 1 0 0 0 0 NA 0 0 5 T_BALL_26 1 1 2 2 19 1 0 0 0 0 NA 0 0 5 T_TALL_1 1 1 2 2 20 1 0 0 0 0 NA 0 0 5 T_TALL_2 1 1 2 2 20 1 0 0 0 0 NA 0 0 5 T_TALL_3 1 1 2 2 20 1 0 0 0 0 NA 0 0 5 T_TALL_4 1 1 2 2 20 1 0 0 0 0 NA 0 0 5 T_TALL_5 1 1 2 2 20 1 0 0 0 0 NA 0 0 5 T_TALL_6 1 1 3 2 20 1 0 0 0 0 NA 0 0 2 T_TALL_7 1 1 3 2 20 1 0 0 0 0 NA 0 0 2 T_TALL_8 1 1 3 2 20 1 0 0 0 0 NA 0 0 10 T_TALL_9 1 1 3 2 20 1 0 0 0 0 NA 0 0 10 T_TALL_10 1 1 3 2 20 1 0 0 0 0 NA 0 0 10 T_TALL_11 1 1 3 2 20 1 0 0 0 0 NA 0 0 10 T_TALL_12 1 1 3 2 20 1 0 0 0 0 NA 0 0 2 T_TALL_13 1 1 3 2 20 1 0 0 0 0 NA 0 0 2 T_TALL_14 1 1 3 2 20 1 0 0 0 0 NA 0 0 2 T_TALL_15 1 1 3 2 20 1 0 0 0 0 NA 0 0 2 T_TALL_16 1 1 3 2 20 1 0 0 0 0 NA 0 0 1 T_TALL_17 1 1 3 2 20 1 0 0 0 0 NA 0 0 1 T_TALL_18 1 1 3 2 20 1 0 0 0 0 NA 0 0 1 TCL_ALLCL_1 1 1 3 3 20 10 0 0 0 0 NA 0 0 10 TCL_ALLCL_2 1 1 3 3 20 10 0 0 0 0 NA 0 0 10 TCL_ALLCL_3 1 1 3 3 20 10 0 0 0 0 NA 0 0 10 TCL_ALLCL_4 1 1 3 3 20 10 0 0 0 0 NA 0 0 10 TCL_ALLCL_5 1 1 3 3 20 10 0 0 0 0 NA 0 0 10 TCL_ALLCL_6 1 1 3 3 20 10 0 0 0 0 NA 0 0 10 TCL_ALLCL_7 1 1 3 3 20 10 0 0 0 0 NA 0 0 10 TCL_ALLCL_8 1 1 3 3 20 10 0 0 0 0 NA 0 0 10 TCL_ALLCL_9 1 1 3 3 20 10 0 0 0 0 NA 0 0 10 TCL_ALLCL_10 1 1 3 3 20 10 0 0 0 0 NA 0 0 10 T_FCC_1 1 1 3 2 21 1 0 0 0 0 NA 0 0 10 T_FCC_2 1 1 3 2 21 1 0 0 0 0 NA 0 0 10 T_FCC_3 1 1 3 2 21 1 0 0 0 0 NA 0 0 10 T_FCC_4 1 1 3 2 21 1 0 0 0 0 NA 0 0 10 T_FCC_5 1 FSCC_S98_14359_(—) MG1999052110AA SR2000060816AA 1 3 2 21 1 0 0 0 0 NA 0 0 10 T_FCC_6 1 1 3 2 21 1 0 0 0 0 NA 0 0 10 T_FCC_7 1 1 3 2 21 1 0 0 0 0 NA 0 0 10 T_FCC_8 1 1 3 2 21 1 0 0 0 0 NA 0 0 10 T_LBL_1 1 1 3 2 22 1 0 0 0 0 NA 0 0 10 T_LBL_2 1 1 3 2 22 1 0 0 0 0 NA 0 0 10 T_LBL_3 1 MG19991001015AA 1 3 2 22 1 0 0 0 0 NA 0 0 10 T_LBL_4 1 1 3 2 22 1 0 0 0 0 NA 0 0 10 T_LBL_5 1 1 3 2 22 1 0 0 0 0 NA 0 0 10 T_LBL_6 1 L_B_CELL_S97_27534_G_(—) MG1999101304AA SR2000060801AA 1 3 2 22 1 0 0 0 0 NA 0 0 10 T_LBL_7 1 1 3 2 22 1 0 0 0 0 NA 0 0 10 T_LBL_8 1 MG1999100110AA 1 3 2 22 1 0 0 0 0 NA 0 0 10 T_MF_1 1 1 4 2 23 1 0 0 0 0 NA 0 0 10 T_MF_2 1 1 4 2 23 1 0 0 0 0 NA 0 0 10 T_MF_3 1 1 4 2 23 1 0 0 0 0 NA 0 0 10 TCL_K562_1 1 1 4 3 24 5 0 0 0 0 NA 0 0 10 TCL_K562_2 1 1 4 3 24 5 0 0 0 0 NA 0 0 10 TCL_HEL_1 1 1 4 3 24 6 0 0 0 0 NA 0 0 10 TCL_HEL_2 1 1 4 3 24 6 0 0 0 0 NA 0 0 10 TCL_HEL_3 1 1 4 3 24 6 0 0 0 0 NA 0 0 10 TCL_TF- 1 1 4 3 24 7 0 0 0 0 NA 0 0 10 1_1 TCL_TF- 1 1 4 3 24 7 0 0 0 0 NA 0 0 10 1_2 TCL_TF- 1 1 4 3 24 7 0 0 0 0 NA 0 0 10 1_3 PDT_BRST_1 2 CUP_5 CL2000080121AA CL2000080818AA 1 1 2 13 1 1 0 1 0 NA 0 0 10 PDT_BRST_2 2 CUP_2 CL2000080117AA CL2000080815AA 1 1 2 13 1 1 0 1 0 NA 0 0 10 PDT_BRST_3 2 CUP_11 CL2000080127AA CL2000080824AA 1 1 2 13 1 1 0 1 0 NA 0 0 10 PDT_BRST_4 2 CUP_3 CL2000080119AA CL2000080816AA 1 1 2 13 1 1 0 1 0 NA 0 0 10 PDT_BRST_5 2 CUP_1 CL2000080118AA CL2000080814AA 1 1 2 13 1 1 0 1 0 NA 0 0 10 PDT_COLON_1 2 CUP_15 CL2000081105AA CL2000081505AA 1 1 2 2 1 1 0 1 1 NA 0 0 10 PDT_LBL_1 2 1 1 2 22 1 2 0 0 0 NA 0 0 10 PDT_LUNG_1 2 CUP_12 CL2000081102AA CL2000081502AA 1 1 2 10 1 1 0 1 0 NA 0 0 10 PDT_LUNG_2 2 CUP_9 CL2000080125AA CL2000080822AA 1 1 2 10 1 1 0 1 0 NA 0 0 10 PDT_LUNG_3 2 CUP_8 CL2000081101AA CL2000081501AA 1 1 2 10 1 1 0 1 0 NA 0 0 10 PDT_LUNG_4 2 CUP_6 CL2000080122AA CL2000080819AA 1 1 2 10 1 1 0 1 0 NA 0 0 10 PDT_LUNG_5 2 CUP_22 CL2000081112AA CL2000081512AA 1 1 2 10 1 1 0 1 0 NA 0 0 10 PDT_LUNG_6 2 CUP_7 CL2000080123AA CL2000080820AA 1 1 2 10 1 1 0 1 0 NA 0 0 10 PDT_LUNG_7 2 CUP_10 CL2000080126AA CL2000080823AA 1 1 2 10 1 1 0 1 0 NA 0 0 10 PDT_LUNG_8 2 CUP_4 CL2000080120AA CL2000080817AA 1 1 2 10 1 1 0 1 0 NA 0 0 10 PDT_OVARY_1 2 CUP_13 CL2000081103AA CL2000081503AA 1 1 2 8 1 1 0 1 0 NA 0 0 10 PDT_OVARY_2 2 CUP_14 CL2000081104AA CL2000081504AA 1 1 2 8 1 1 0 1 0 NA 0 0 10 PDT_OVARY_3 2 CUP_17 CL2000081107AA CL2000081507AA 1 1 2 8 1 1 0 1 0 NA 0 0 10 PDT_STOM_1 2 1 1 2 1 1 2 0 1 1 NA 0 0 10 N_MLUNG_1 3 1 4 1 26 1 0 0 1 0 NA 0 0 5 N_MLUNG_2 3 1 4 1 26 1 0 0 1 0 NA 0 0 5 N_MLUNG_3 3 1 4 1 26 1 0 0 1 0 NA 0 0 5 N_MLUNG_4 3 1 4 1 26 1 0 0 1 0 NA 0 0 5 N_MLUNG_5 3 1 4 1 26 1 0 0 1 0 NA 0 0 5 T_MLUNG_1 3 1 4 2 26 1 0 0 1 0 NA 0 0 5 T_MLUNG_2 3 1 4 2 26 1 0 0 1 0 NA 0 0 5 T_MLUNG_3 3 1 4 2 26 1 0 0 1 0 NA 0 0 5 T_MLUNG_4 3 1 4 2 26 1 0 0 1 0 NA 0 0 5 T_MLUNG_5 3 1 4 2 26 1 0 0 1 0 NA 0 0 5 T_MLUNG_6 3 1 4 2 26 1 0 0 1 0 NA 0 0 5 T_MLUNG_7 3 1 4 2 26 1 0 0 1 0 NA 0 0 5 T_SJ_ALL_1 4 2 2 2 19 1 0 3 0 0 NA 0 0 5 T_SJ_ALL_2 4 2 2 2 19 1 0 9 0 0 NA 0 0 5 T_SJ_ALL_3 4 2 2 2 19 1 0 4 0 0 NA 0 0 5 T_SJ_ALL_4 4 2 2 2 19 1 0 3 0 0 NA 0 0 5 T_SJ_ALL_5 4 2 2 2 20 1 0 6 0 0 NA 0 0 5 T_SJ_ALL_6 4 2 2 2 19 1 0 1 0 0 NA 0 0 5 T_SJ_ALL_7 4 2 2 2 19 1 0 9 0 0 NA 0 0 5 T_SJ_ALL_8 4 2 2 2 20 1 0 6 0 0 NA 0 0 5 T_SJ_ALL_9 4 2 2 2 19 1 0 4 0 0 NA 0 0 5 T_SJ_ALL_10 4 2 2 2 19 1 0 5 0 0 NA 0 0 5 T_SJ_ALL_11 4 2 2 2 19 1 0 7 0 0 NA 0 0 5 T_SJ_ALL_12 4 2 2 2 19 1 0 7 0 0 NA 0 0 5 T_SJ_ALL_13 4 2 2 2 19 1 0 7 0 0 NA 0 0 5 T_SJ_ALL_14 4 2 2 2 20 1 0 6 0 0 NA 0 0 5 T_SJ_ALL_15 4 2 2 2 19 1 0 3 0 0 NA 0 0 5 T_SJ_ALL_16 4 2 2 2 19 1 0 4 0 0 NA 0 0 5 T_SJ_ALL_17 4 2 2 2 20 1 0 6 0 0 NA 0 0 5 T_SJ_ALL_18 4 2 2 2 20 1 0 6 0 0 NA 0 0 5 T_SJ_ALL_19 4 2 2 2 19 1 0 9 0 0 NA 0 0 5 T_SJ_ALL_20 4 2 2 2 19 1 0 7 0 0 NA 0 0 5 T_SJ_ALL_21 4 2 2 2 20 1 0 6 0 0 NA 0 0 5 T_SJ_ALL_22 4 2 2 2 20 1 0 6 0 0 NA 0 0 5 T_SJ_ALL_23 4 2 2 2 19 1 0 9 0 0 NA 0 0 5 T_SJ_ALL_24 4 2 2 2 20 1 0 6 0 0 NA 0 0 5 T_SJ_ALL_25 4 2 2 2 19 1 0 5 0 0 NA 0 0 5 T_SJ_ALL_26 4 2 2 2 20 1 0 6 0 0 NA 0 0 5 T_SJ_ALL_27 4 2 2 2 19 1 0 2 0 0 NA 0 0 5 T_SJ_ALL_28 4 2 2 2 19 1 0 4 0 0 NA 0 0 5 T_SJ_ALL_29 4 2 2 2 19 1 0 2 0 0 NA 0 0 5 T_SJ_ALL_30 4 2 2 2 19 1 0 1 0 0 NA 0 0 5 T_SJ_ALL_31 4 2 2 2 19 1 0 5 0 0 NA 0 0 5 T_SJ_ALL_32 4 2 2 2 19 1 0 5 0 0 NA 0 0 5 T_SJ_ALL_33 4 2 2 2 19 1 0 1 0 0 NA 0 0 5 T_SJ_ALL_34 4 2 2 2 20 1 0 6 0 0 NA 0 0 5 T_SJ_ALL_35 4 2 2 2 19 1 0 2 0 0 NA 0 0 5 T_SJ_ALL_36 4 2 2 2 20 1 0 6 0 0 NA 0 0 5 T_SJ_ALL_37 4 2 2 2 20 1 0 6 0 0 NA 0 0 5 T_SJ_ALL_38 4 2 2 2 19 1 0 3 0 0 NA 0 0 5 T_SJ_ALL_39 4 2 2 2 19 1 0 4 0 0 NA 0 0 5 T_SJ_ALL_40 4 2 2 2 20 1 0 6 0 0 NA 0 0 5 T_SJ_ALL_41 4 2 2 2 19 1 0 2 0 0 NA 0 0 5 T_SJ_ALL_42 4 2 2 2 19 1 0 7 0 0 NA 0 0 5 T_SJ_ALL_43 4 2 2 2 20 1 0 6 0 0 NA 0 0 5 T_SJ_ALL_44 4 2 2 2 19 1 0 3 0 0 NA 0 0 5 T_SJ_ALL_45 4 2 2 2 19 1 0 4 0 0 NA 0 0 5 T_SJ_ALL_46 4 2 2 2 19 1 0 7 0 0 NA 0 0 5 T_SJ_ALL_47 4 2 2 2 20 1 0 6 0 0 NA 0 0 5 T_SJ_ALL_48 4 2 2 2 19 1 0 1 0 0 NA 0 0 5 T_SJ_ALL_49 4 2 2 2 19 1 0 5 0 0 NA 0 0 5 T_SJ_ALL_50 4 2 2 2 19 1 0 4 0 0 NA 0 0 5 T_SJ_ALL_51 4 2 2 2 19 1 0 3 0 0 NA 0 0 5 T_SJ_ALL_52 4 2 2 2 19 1 0 3 0 0 NA 0 0 5 T_SJ_ALL_53 4 2 2 2 19 1 0 5 0 0 NA 0 0 5 T_SJ_ALL_54 4 2 2 2 20 1 0 6 0 0 NA 0 0 5 T_SJ_ALL_55 4 2 2 2 20 1 0 6 0 0 NA 0 0 5 T_SJ_ALL_56 4 2 2 2 19 1 0 4 0 0 NA 0 0 5 T_SJ_ALL_57 4 2 2 2 19 1 0 2 0 0 NA 0 0 5 T_SJ_ALL_58 4 2 2 2 20 1 0 6 0 0 NA 0 0 5 T_SJ_ALL_59 4 2 2 2 20 1 0 6 0 0 NA 0 0 5 T_SJ_ALL_60 4 2 2 2 19 1 0 7 0 0 NA 0 0 5 T_SJ_ALL_61 4 2 2 2 19 1 0 7 0 0 NA 0 0 5 T_SJ_ALL_62 4 2 2 2 19 1 0 3 0 0 NA 0 0 5 T_SJ_ALL_63 4 2 2 2 19 1 0 7 0 0 NA 0 0 5 T_SJ_ALL_64 4 2 2 2 19 1 0 2 0 0 NA 0 0 5 T_SJ_ALL_65 4 2 2 2 19 1 0 2 0 0 NA 0 0 5 T_SJ_ALL_66 4 2 2 2 19 1 0 2 0 0 NA 0 0 5 T_SJ_ALL_67 4 2 2 2 19 1 0 1 0 0 NA 0 0 5 T_SJ_ALL_68 4 2 2 2 19 1 0 7 0 0 NA 0 0 5 T_SJ_ALL_69 4 2 2 2 19 1 0 2 0 0 NA 0 0 5 T_SJ_ALL_70 4 2 2 2 19 1 0 4 0 0 NA 0 0 5 T_SJ_ALL_71 4 2 2 2 19 1 0 3 0 0 NA 0 0 5 T_SJ_ALL_72 4 2 2 2 19 1 0 7 0 0 NA 0 0 5 T_SJ_ALL_73 4 2 2 2 19 1 0 3 0 0 NA 0 0 5 TCL_HL60_1 5 1 4 3 24 9 0 0 0 0 1- 0 0 5 Day − ATRA TCL_HL60_2 5 1 4 3 24 9 0 0 0 0 3- 0 0 5 Day − ATRA TCL_HL60_3 5 1 4 3 24 9 0 0 0 0 5- 0 0 5 Day − ATRA TCL_HL60_4 5 1 4 3 24 9 0 0 0 0 1- 0 0 5 Day + ATRA TCL_HL60_5 5 1 4 3 24 9 0 0 0 0 3- 0 0 5 Day + ATRA TCL_HL60_6 5 1 4 3 24 9 0 0 0 0 5- 0 0 5 Day + ATRA N_ERYTH_1 6 2 4 1 27 1 0 0 0 0 2- 0 0 1.6 Day N_ERYTH_2 6 2 4 1 27 1 0 0 0 0 4- 0 0 1.6 Day N_ERYTH_3 6 2 4 1 27 1 0 0 0 0 6- 0 0 1.6 Day N_ERYTH_4 6 2 4 1 27 1 0 0 0 0 8- 0 0 1.6 Day N_ERYTH_5 6 2 4 1 27 1 0 0 0 0 10- 0 0 1.6 Day N_ERYTH_6 6 2 4 1 27 1 0 0 0 0 12- 0 0 1.6 Day

Table 10a-10b

Probe Information Field Description Probe ID Probe name Seq Type Biosequence type; oligo for deoxyoligonucleotides Probe 5′ to 3′ capture probe sequence Sequence Target 5′ to 3′ target or target mutant sequence; NA for not Sequence available Human Human miRNA recognized by probe according to microRNA registry rfam 5.0 Mouse Mouse miRNA recognized by probe according to microRNA registry rfam 5.0 Rat Rat miRNA recognized by probe according to microRNA registry rfam 5.0 Other Special note about recognition Control Whether the feature is a control feature and what type of control Set Number The set of beads this feature belongs to in version 1 (V1) Set Number The set of beads this feature belongs to in version 2 (V2) Usage Whether the feature is used in the final dataset for analyses and why not

TABLE 10a Probe Seq ID Type Probe Sequence Target Sequence EAM103 Oligo /5AmMC6/TGGCATTCACCGCGTGCCTTA UUAAGGCACGCGGUGAAUGCCA seq id no: 286 seq id no: 568 EAM105 Oligo /5AmMC6/TCACAAGTTAGGGTCTCAGGGA UCCCUGAGACCCUAACUUGUGA seq id no: 287 seq id no: 569 EAM109 Oligo /5AmMC6/AACAACAAAATCACTAGTCTTCCA UGGAAGACUAGUGAUUUUGUU seq id no: 288 seq id no: 570 EAM111 Oligo /5AmMC6/TAACTGTACAAACTACTACCTCA UGAGGUAGUAGUUUGUACAGU seq id no: 289 seq id no: 571 EAM115 Oligo /5AmMC6/CGCCAATATTTACGTGCTGCTA UAGCAGCACGUAAAUAUUGGCG seq id no: 290 seq id no: 572 EAM119 Oligo /5AmMC6/AACACTGATTTCAAATGGTGCTA UAGCACCAUUUGAAAUCAGUGU seq id no: 291 seq id no: 573 EAM121 Oligo /5AmMC6/CACAAGATCGGATCTACGGGT AACCCGUAGAUCCGAUCUUGUG seq id no: 292 seq id no: 574 EAM131 Oligo /5AmMC6/ACAGGCCGGGACAAGTGCAATAT UAUUGCACUUGUCCCGGCCUGU seq id no: 293 seq id no: 575 EAM139 Oligo /5AmMC6/TAACCCATGGAATTCAGTTCTCA UGAGAACUGAAUUCCAUGGGUU seq id no: 294 seq id no: 576 EAM145 Oligo /5AmMC6/AACCATACAACCTACTACCTCA UGAGGUAGUAGGUUGUAUGGUU seq id no: 295 seq id no: 577 EAM152 Oligo /5AmMC6/ACTTTCGGTTATCTAGCTTTAT UAAAGCUAGAUAACCGAAAGU seq id no: 296 seq id no: 578 EAM238 Oligo /5AmMC6/ATACATACTTCTTTACATTCCA UGGAAUGUAAAGAAGUAUGUA seq id no: 297 seq id no: 579 EAM270 Oligo /5AmMC6/GCTGAGTGTAGGATGTTTACA UGUAAACAUCCUACACUCAGC seq id no: 298 seq id no: 580 EAM159 Oligo /5AmMC6/ATGCCCTTTTAACATTGCACTG CAGUGCAAUGUUAAAAGGGC seq id no: 299 seq id no: 581 EAM163 Oligo /5AmMC6/TCCATAAAGTAGGAAACACTACA UGUAGUGUUUCCUACUUUAUGGA seq id no: 300 seq id no: 582 EAM171 Oligo /5AmMC6/CTACGCGTATTCTTAAGCAATAA UAUUGCUUAAGAAUACGCGUAG seq id no: 301 seq id no: 583 EAM183 Oligo /5AmMC6/AGCACAAACTACTACCTCA UGAGGUAGUAGUUUGUGCU seq id no: 302 seq id no: 584 EAM184 Oligo /5AmMC6/CACAAGTTCGGATCTACGGGTT AACCCGUAGAUCCGAACUUGUG seq id no: 303 seq id no: 585 EAM186 Oligo /5AmMC6/GCTACCTGCACTGTAAGCACTTTT AAAAGUGCUUACAGUGCAGGUAGC seq id no: 304 seq id no: 586 EAM189 Oligo /5AmMC6/CACAAATTCGGATCTACAGGGTA UACCCUGUAGAUCCGAAUUUGUG seq id no: 305 seq id no: 587 EAM191 Oligo /5AmMC6/ACAAACACCATTGTCACACTCCA UGGAGUGUGACAAUGGUGUUUGU seq id no: 306 seq id no: 588 EAM192 Oligo /5AmMC6/CGCGTACCAAAAGTAATAATG CAUUAUUACUUUUGGUACGCG seq id no: 307 seq id no: 589 EAM198 Oligo /5AmMC6/GCCCTTTCATCATTGCACTG CAGUGCAAUGAUGAAAGGGCAU seq id no: 308 seq id no: 590 EAM202 Oligo /5AmMC6/TCCCTCTGGTCAACCAGTCACA UGUGACUGGUUGACCAGAGGG seq id no: 309 seq id no: 591 EAM209 Oligo /5AmMC6/GTAGTGCTTTCTACTTTATG CAUAAAGUAGAAAGCACUAC seq id no: 310 seq id no: 592 EAM221 Oligo /5AmMC6/CCCCTATCACAATTAGCATTAA UUAAUGCUAAUUGUGAUAGGGG seq id no: 311 seq id no: 593 EAM223 Oligo /5AmMC6/TGTAAACCATGATGTGCTGCTA UAGCAGCACAUCAUGGUUUACA seq id no: 312 seq id no: 594 EAM224 Oligo /5AmMC6/ACTACCTGCACTGTAAGCACTTTG CAAAGUGCUUACAGUGCAGGUAGU seq id no: 313 seq id no: 595 EAM225 Oligo /5AmMC6/TATCTGCACTAGATGCACCTTA UAAGGUGCAUCUAGUGCAGAUA seq id no: 314 seq id no: 596 EAM226 Oligo /5AmMC6/ACTCACCGACAGCGTTGAATGTT AACAUUCAACGCUGUCGGUGAGU seq id no: 315 seq id no: 597 EAM227 Oligo /5AmMC6/AACCCACCGACAGCAATGAATGTT AACAUUCAUUGCUGUCGGUGGGUU seq id no: 316 seq id no: 598 EAM234 Oligo /5AmMC6/GAACAGGTAGTCTGAACACTGGG CCCAGUGUUCAGACUACCUGUUC seq id no: 317 seq id no: 599 EAM235 Oligo /5AmMC6/GAACAGATAGTCTAAACACTGGG CCCAGUGUUUAGACUAUCUGUUC seq id no: 318 seq id no: 600 EAM236 Oligo /5AmMC6/TCAGTTTTGCATAGATTTGCACA UGUGCAAAUCUAUGCAAAACUGA seq id no: 319 seq id no: 601 EAM241 Oligo /5AmMC6/CTAGTGGTCCTAAACATTTCAC GUGAAAUGUUUAGGACCACUAG seq id no: 320 seq id no: 602 EAM242 Oligo /5AmMC6/AGGCATAGGATGACAAAGGGAA UUCCCUUUGUCAUCCUAUGCCUG seq id no: 321 seq id no: 603 EAM243 Oligo /5AmMC6/CAGACTCCGGTGGAATGAAGGA UCCUUCAUUCCACCGGAGUCUG seq id no: 322 seq id no: 604 EAM245 Oligo /5AmMC6/CAGCCGCTGTCACACGCACAG CUGUGCGUGUGACAGCGGCUG seq id no: 323 seq id no: 605 EAM249 Oligo /5AmMC6/CTGCCTGTCTGTGCCTGCTGT ACAGCAGGCACAGACAGGCAG seq id no: 324 seq id no: 606 EAM254 Oligo /5AmMC6/AGAATTGCGTTTGGACAATCA UGAUUGUCCAAACGCAAUUCU seq id no: 325 seq id no: 607 EAM257 Oligo /5AmMC6/GAAACCCAGCAGACAATGTAGCT AGCUACAUUGUCUGCUGGGUUUC seq id no: 326 seq id no: 608 EAM258 Oligo /5AmMC6/GAGACCCAGTAGCCAGATGTAGCT AGCUACAUCUGGCUACUGGGUCUC seq id no: 327 seq id no: 609 EAM259 Oligo /5AmMC6/GGGGTATTTGACAAACTGACA UGUCAGUUUGUCAAAUACCCC seq id no: 328 seq id no: 610 EAM273 Oligo /5AmMC6/CAATGCAACTACAATGCAC GUGCAUUGUAGUUGCAUUG seq id no: 329 seq id no: 611 EAM288 Oligo /5AmMC6/ACACAAATTCGGTTCTACAGGG CCCUGUAGAACCGAAUUUGUGU seq id no: 330 seq id no: 612 EAM293 Oligo /5AmMC6/ACCCTCCACCATGCAAGGGATG CAUCCCUUGCAUGGUGGAGGGU seq id no: 331 seq id no: 613 EAM297 Oligo /5AmMC6/CTGGGACTTTGTAGGCCAGTT AACUGGCCUACAAAGUCCCAG seq id no: 332 seq id no: 614 EAM301 Oligo /5AmMC6/CCTATCTCCCCTCTGGACC GGUCCAGAGGGGAGAUAGG seq id no: 333 seq id no: 615 EAM304 Oligo /5AmMC6/CATCGTTACCAGACAGTGTTA UAACACUGUCUGGUAACGAUGU seq id no: 334 seq id no: 616 EAM306 Oligo /5AmMC6/AGAACAATGCCTTACTGAGTA UACUCAGUAAGGCAUUGUUCU seq id no: 335 seq id no: 617 EAM307 Oligo /5AmMC6/TCTTCCCATGCGCTATACCTCT AGAGGUAUAGCGCAUGGGAAGA seq id no: 336 seq id no: 618 EAM308 Oligo /5AmMC6/CCACACACTTCCTTACATTCCA UGGAAUGUAAGGAAGUGUGUGG seq id no: 337 seq id no: 619 EAM309 Oligo /5AmMC6/GAGGGAGGAGAGCCAGGAGAAGC GCUUCUCCUGGCUCUCCUCCCUC seq id no: 338 seq id no: 620 EAM310 Oligo /5AmMC6/ACAAGCTTTTTGCTCGTCTTAT AUAAGACGAGCAAAAAGCUUGU seq id no: 339 seq id no: 621 EAM247 Oligo /5AmMC6/GGCCGTGACTGGAGACTGTTA UAACAGUCUCCAGUCACGGCC seq id no: 340 seq id no: 622 EAM251 Oligo /5AmMC6/CACAGTTGCCAGCTGAGATTA UAAUCUCAGCUGGCAACUGUG seq id no: 341 seq id no: 623 EAM253 Oligo /5AmMC6/ACATGGTTAGATCAAGCACAA UUGUGCUUGAUCUAACCAUGU seq id no: 342 seq id no: 624 EAM275 Oligo /5AmMC6/ACAACCAGCTAAGACACTGCCA UGGCAGUGUCUUAGCUGGUUGUU seq id no: 343 seq id no: 625 EAM246 Oligo /5AmMC6/AGGCGAAGGATGACAAAGGGAA UUCCCUUUGUCAUCCUUCGCCU seq id no: 344 seq id no: 626 EAM250 Oligo /5AmMC6/GTCTGTCAATTCATAGGTCAT AUGACCUAUGAAUUGACAGAC seq id no: 345 seq id no: 627 EAM252 Oligo /5AmMC6/ATCCAATCAGTTCCTGATGCAGTA UACUGCAUCAGGAACUGAUUGGAU seq id no: 346 seq id no: 628 EAM305 Oligo /5AmMC6/GTCATCATTACCAGGCAGTATTA UAAUACUGCCUGGUAAUGAUGAC seq id no: 347 seq id no: 629 EAM303 Oligo /5AmMC6/AACCAATGTGCAGACTACTGTA UACAGUAGUCUGCACAUUGGUU seq id no: 348 seq id no: 630 EAM300 Oligo /5AmMC6/GCTGGGTGGAGAAGGTGGTGAA UUCACCACCUUCUCCACCCAGC seq id no: 349 seq id no: 631 EAM299 Oligo /5AmMC6/GCCAATATTTCTGTGCTGCTA UAGCAGCACAGAAAUAUUGGC seq id no: 350 seq id no: 632 EAM298 Oligo /5AmMC6/TCCACATGGAGTTGCTGTTACA UGUAACAGCAACUCCAUGUGGA seq id no: 351 seq id no: 633 EAM296 Oligo /5AmMC6/AGCTGCTTTTGGGATTCCGTTG CAACGGAAUCCCAAAAGCAGCU seq id no: 352 seq id no: 634 EAM295 Oligo /5AmMC6/ACCTAATATATCAAACATATCA UGAUAUGUUUGAUAUAUUAGGU seq id no: 353 seq id no: 635 EAM292 Oligo /5AmMC6/AAGCCCAAAAGGAGAATTCTTTG CAAAGAAUUCUCCUUUUGGGCUU seq id no: 354 seq id no: 636 EAM112 Oligo /5AmMC6/TAACTGTAGAAAGTACTACCTCA TGAGGTAGTACTTTCTACAGTTA seq id no: 355 seq id no: 637 EAM116 Oligo /5AmMC6/CGCCAATATTAAGGTGCTGCTA TAGCAGCACCTTAATATTGGCG seq id no: 356 seq id no:638 EAM120 Oligo /5AmMC6/AACACTGATTTGAAAAGGTGCTA TAGCACCTTTTCAAATCAGTGTT seq id no: 357 seq id no: 639 EAM122 Oligo /5AmMC6/CACAAGATGGGATGTACGGGT ACCCGTACATCCCATCTTGTG seq id no: 358 seq id no: 640 EAM132 Oligo /5AmMC6/ACAGGCCGGGAGAAGAGCAATAT ATATTGCTCTTCTCCCGGCCTGT seq id no: 359 seq id no: 641 EAM140 Oligo /5AmMC6/TAACCCATGGAAATGAGTTCTCA TGAGAACTCATTTCCATGGGTTA seq id no: 360 seq id no: 642 EAM282 Oligo /5AmMC6/GAACAGGTAGTCTAAACACTGGG CCCAGUGUUUAGACUACCUGUUC seq id no: 361 seq id no: 643 EAM281 Oligo /5AmMC6/atccagtcagttcctgatgcagta UACUGCAUCAGGAACUGACUGGAU seq id no: 362 seq id no: 644 EAM280 Oligo /5AmMC6/GCTGCAAACATCCGACTGAAAG CUUUCAGUCGGAUGUUUGCAGC seq id no: 363 seq id no: 645 EAM279 Oligo /5AmMC6/TAACCGATTTCAAATGGTGCTA UAGCACCAUUUGAAAUCGGUUA seq id no: 364 seq id no: 646 EAM278 Oligo /5AmMC6/AACAATACAACTTACTACCTCA UGAGGUAGUAAGUUGUAUUGUU seq id no: 365 seq id no: 647 EAM277 Oligo /5AmMC6/GCAAAAATGTGCTAGTGCCAAA UUUGGCACUAGCACAUUUUUGCU seq id no: 366 seq id no: 648 EAM276 Oligo /5AmMC6/TCATACAGCTAGATAACCAAAGA UCUUUGGUUAUCUAGCUGUAUGA seq id no: 367 seq id no: 649 EAM272 Oligo /5AmMC6/CTTCCAGTCGGGGATGTTTACA UGUAAACAUCCCCGACUGGAAG seq id no: 368 seq id no: 650 EAM271 Oligo /5AmMC6/GCTGAGAGTGTAGGATGTTTACA UGUAAACAUCCUACACUCUCAGC seq id no: 369 seq id no: 651 EAM268 Oligo /5AmMC6/AACCGATTTCAGATGGTGCTAG CUAGCACCAUCUGAAAUCGGUU seq id no: 370  seq id no: 652 EAM264 Oligo /5AmMC6/CAGAACTTAGCCACTGTGAA UUCACAGUGGCUAAGUUCUG seq id no: 371 seq id no: 653 EAM263 Oligo /5AmMC6/AGCCTATCCTGGATTACTTGAA UUCAAGUAAUCCAGGAUAGGCU seq id no: 372 seq id no: 654 EAM262 Oligo /5AmMC6/CTGTTCCTGCTGAACTGAGCCA UGGCUCAGUUCAGCAGGAACAG seq id no: 373 seq id no: 655 EAM261 Oligo /5AmMC6/GTGGTAATCCCTGGCAATGTGAT AUCACAUUGCCAGGGAUUACCAC seq id no: 374 seq id no: 656 EAM260 Oligo /5AmMC6/GGAAATCCCTGGCAATGTGAT AUCACAUUGCCAGGGAUUUCC seq id no: 375 seq id no: 657 EAM256 Oligo /5AmMC6/AAAGTGTCAGATACGGTGTGG CCACACCGUAUCUGACACUUU seq id no: 376 seq id no: 658 EAM255 Oligo /5AmMC6/ACAGTTCTTCAACTGGCAGCTT AAGCUGCCAGUUGAAGAACUGU seq id no: 377 seq id no: 659 EAM248 Oligo /5AmMC6/GGTACAATCAACGGTCGATGGT ACCAUCGACCGUUGAUUGUACC seq id no: 378 seq id no: 660 EAM244 Oligo /5AmMC6/TCAACATCAGTCTGATAAGCTA UAGCUUAUCAGACUGAUGUUGA seq id no: 379 seq id no: 661 EAM240 Oligo /5AmMC6/CTACCTGCACTATAAGCACTTTA UAAAGUGCUUAUAGUGCAGGUAG seq id no: 380 seq id no: 662 EAM237 Oligo /5AmMC6/TCAGTTTTGCATGGATTTGCACA UGUGCAAAUCCAUGCAAAACUGA seq id no: 381 seq id no: 663 EAM233 Oligo /5AmMC6/CCCAACAACATGAAACTACCTA UAGGUAGUUUCAUGUUGUUGG seq id no: 382 seq id no: 664 EAM232 Oligo /5AmMC6/GGCTGTCAATTCATAGGTCAG CUGACCUAUGAAUUGACAGCC seq id no: 383 seq id no: 665 EAM231 Oligo /5AmMC6/CGGCTGCAACACAAGACACGA UCGUGUCUUGUGUUGCAGCCGG seq id no: 384 seq id no: 666 EAM230 Oligo /5AmMC6/CAGTGAATTCTACCAGTGCCATA UAUGGCACUGGUAGAAUUCACUG seq id no: 385 seq id no: 667 EAM229 Oligo /5AmMC6/TGTGAGTTCTACCATTGCCAAA UUUGGCAAUGGUAGAACUCACA seq id no: 386 seq id no: 668 EAM228 Oligo /5AmMC6/ACTCACCGACAGGTTGAATGTT AACAUUCAACCUGUCGGUGAGU seq id no: 387 seq id no: 669 EAM222 Oligo /5AmMC6/CACAAACCATTATGTGCTGCTA UAGCAGCACAUAAUGGUUUGUG seq id no: 388 seq id no: 670 EAM220 Oligo /5AmMC6/CGAAGGCAACACGGATAACCTA UAGGUUAUCCGUGUUGCCUUCG seq id no: 389 seq id no: 671 EAM219 Oligo /5AmMC6/TCACTTTTGTGACTATGCAA UUGCAUAGUCACAAAAGUGA seq id no: 390 seq id no: 672 EAM218 Oligo /5AmMC6/CCAAGTTCTGTCATGCACTGA UCAGUGCAUGACAGAACUUGG seq id no: 391 seq id no: 673 EAM217 Oligo /5AmMC6/ACACTGGTACAAGGGTTGGGAGA UCUCCCAACCCUUGUACCAGUG seq id no: 392 seq id no: 674 EAM216 Oligo /5AmMC6/GGAGTGAAGACACGGAGCCAGA UCUGGCUCCGUGUCUUCACUCC seq id no: 393 seq id no: 675 EAM215 Oligo /5AmMC6/ACAAAGTTCTGTGATGCACTGA UCAGUGCAUCACAGAACUUUGU seq id no: 394 seq id no: 676 EAM214 Oligo /5AmMC6/ACAAAGTTCTGTAGTGCACTGA UCAGUGCACUACAGAACUUUGU seq id no: 395 seq id no: 677 EAM212 Oligo /5AmMC6/AAGGGATTCCTGGGAAAACTGGAC GUCCAGUUUUCCCAGGAAUCCCUU seq id no: 396 seq id no: 678 EAM211 Oligo /5AmMC6/CTAGTACATCATCTATACTGTA UACAGUAUAGAUGAUGUACUAG seq id no: 397 seq id no: 679 EAM210 Oligo /5AmMC6/tgAGCTACAGTGCTTCATCTCA UGAGAUGAAGCACUGUAGCUCA seq id no: 398 seq id no: 680 EAM208 Oligo /5AmMC6/CCATCTTTACCAGACAGTGTT AACACUGUCUGGUAAAGAUGG seq id no: 399 seq id no: 681 EAM207 Oligo /5AmMC6/CTACCATAGGGTAAAACCACT AGUGGUUUUACCCUAUGGUAG seq id no: 400 seq id no: 682 EAM206 Oligo /5AmMC6/AGACACGTGCACTGTAGA UCUACAGUGCACGUGUCU seq id no: 401 seq id no: 683 EAM205 Oligo /5AmMC6/GATTCACAACACCAGCT AGCUGGUGUUGUGAAUC seq id no: 402 seq id no: 684 EAM203 Oligo /5AmMC6/TTCACATAGGAATAAAAAGCCATA UAUGGCUUUUUAUUCCUAUGUGA seq id no: 403 seq id no: 685 EAM200 Oligo /5AmMC6/ACAGCTGGTTGAAGGGGACCAA UUGGUCCCCUUCAACCAGCUGU seq id no: 404 seq id no: 686 EAM195 Oligo /5AmMC6/GAAAGAGACCGGTTCACTGTGA UCACAGUGAACCGGUCUCUUUC seq id no: 405 seq id no: 687 EAM194 Oligo /5AmMC6/AAAAGAGACCGGTTCACTGTGA UCACAGUGAACCGGUCUCUUUU seq id no: 406 seq id no: 688 EAM193 Oligo /5AmMC6/CACAGGTTAAAGGGTCTCAGGGA UCCCUGAGACCCUUUAACCUGUG seq id no: 407 seq id no: 689 EAM190 Oligo /5AmMC6/ACAAATTCGGTTCTACAGGGTA UACCCUGUAGAACCGAAUUUGU seq id no: 408 seq id no: 690 EAM187 Oligo /5AmMC6/TGATAGCCCTGTACAATGCTGCT AGCAGCAUUGUACAGGGCUAUCA seq id no: 409 seq id no: 691 EAM185 Oligo /5AmMC6/TCATAGCCCTGTACAATGCTGCT AGCAGCAUUGUACAGGGCUAUGA seq id no: 410 seq id no: 692 EAM181 Oligo /5AmMC6/AACTATACAATCTACTACCTCA UGAGGUAGUAGAUUGUAUAGUU seq id no: 411 seq id no: 693 EAM179 Oligo /5AmMC6/ACTATGCAACCTACTACCTCT AGAGGUAGUAGGUUGCAUAGU seq id no: 412 seq id no: 694 EAM177 Oligo /5AmMC6/TTCAGCTATCACAGTACTGTA UACAGUACUGUGAUAGCUGAAG seq id no: 413 seq id no: 695 EAM175 Oligo /5AmMC6/TCGCCCTCTCAACCCAGCTTTT AAAAGCUGGGUUGAGAGGGCGAA seq id no: 414 seq id no: 696 EAM168 Oligo /5AmMC6/CTATACAACCTCCTACCTCA UGAGGUAGGAGGUUGUAUAGU seq id no: 415 seq id no: 697 EAM161 Oligo /5AmMC6/CTCAATAGACTGTGAGCTCCTT AAGGAGCUCACAGUCUAUUGAG seq id no: 416 seq id no: 698 EAM160 Oligo /5AmMC6/AACCTATCCTGAATTACTTGAA UUCAAGUAAUUCAGGAUAGGUU seq id no: 417 seq id no: 699 EAM155 Oligo /5AmMC6/TCCATCATCAAAACAAATGGAGT ACUCCAUUUGUUUUGAUGAUGGA seq id no: 418 seq id no: 700 EAM153 Oligo /5AmMC6/AACTATACAACCTACTACCTCA UGAGGUAGUAGGUUGUAUAGUU seq id no: 419 seq id no: 701 EAM147 Oligo /5AmMC6/AACCACACAACCTACTACCTCA UGAGGUAGUAGGUUGUGUGGUU seq id no: 420 seq id no: 702 EAM137 Oligo /5AmMC6/CCGACCATGGCTGTAGACTGTTA UAACAGUCUACAGCCAUGGUCG seq id no: 421 seq id no: 703 EAM133 Oligo /5AmMC6/ACACCAATGCCCTAGGGGATGCG CGCAUCCCCUAGGGCAUUGGUGU seq id no: 422 seq id no: 704 EAM311 Oligo /5AmMC6/CTTCAGTTATCACAGTACTGTA UACAGUACUGUGAUAACUGAAG seq id no: 423 seq id no: 705 EAM312 Oligo /5AmMC6/ACAGGAGTCTGAGCATTTGA UCAAAUGCUCAGACUCCUGU seq id no: 424 seq id no: 706 EAM313 Oligo /5AmMC6/ATCTGCACTGTCAGCACTTTA UAAAGUGCUGACAGUGCAGAU seq id no: 425 seq id no: 707 EAM314 Oligo /5AmMC6/GCATTATTACTCACGGTACGA UCGUACCGUGAGUAAUAAUGC seq id no: 426 seq id no: 708 EAM315 Oligo /5AmMC6/AGCCAAGCTCAGACGGATCCGA UCGGAUCCGUCUGAGCUUGGCU seq id no: 427 seq id no: 709 EAM316 Oligo /5AmMC6/GCAGAAGCATTTCCACACAC GUGUGUGGAAAUGCUUCUGC seq id no: 428 seq id no: 710 EAM317 Oligo /5AmMC6/CCCCTATCACGATTAGCATTAA UUAAUGCUAAUCGUGAUAGGGG seq id no: 429 seq id no: 711 EAM318 Oligo /5AmMC6/ACAAGTGCCTTCACTGCAGT ACUGCAGUGAAGGCACUUGU seq id no: 430 seq id no: 712 EAM319 Oligo /5AmMC6/TAGTTGGCAAGTCTAGAACCA UGGUUCUAGACUUGCCAACUA seq id no: 431 seq id no: 713 EAM320 Oligo /5AmMC6/ACTGATATCAGCTCAGTAGGCAC GUGCCUACUGAGCUGAUAUCAGU seq id no: 432 seq id no: 714 EAM321 Oligo /5AmMC6/CATCATTACCAGGCAGTATTAGA CUCUAAUACUGCCUGGUAAUGAUG seq id no: 433 seq id no: 715 EAM291 Oligo /5AmMC6/GAACTGCCTTTCTCTCCA UGGAGAGAAAGGCAGUUC seq id no: 434 seq id no: 716 EAM290 Oligo /5AmMC6/ACCCTTATCAGTTCTCCGTCCA UGGACGGAGAACUGAUAAGGGU seq id no: 435 seq id no: 717 EAM322 Oligo /5AmMC6/TCCATCATTACCCGGCAGTATT AAUACUGCCGGGUAAUGAUGGA seq id no: 436 seq id no: 718 EAM323 Oligo /5AmMC6/TAAACGGAACCACTAGTGACTTG CAAGUCACUAGUGGUUCCGUUUA seq id no: 437 seq id no: 719 EAM324 Oligo /5AmMC6/TCAGACCGAGACAAGTGCAATG CAUUGCACUUGUCUCGGUCUGA seq id no: 438 seq id no: 720 EAM325 Oligo /5AmMC6/GGCGGAACTTAGCCACTGTGAA UUCACAGUGGCUAAGUUCCGCC seq id no: 439 seq id no: 721 EAM326 Oligo /5AmMC6/ACAGGATTGAGGGGGGGCCCT AGGGCCCCCCCUCAAUCCUGU seq id no: 440 seq id no: 722 EAM327 Oligo /5AmMC6/ATGTATGTGGGACGGTAAACCA UGGUUUACCGUCCCACAUACAU seq id no: 441 seq id no: 723 EAM328 Oligo /5AmMC6/GCTTTGACAATACTATTGCACTG CAGUGCAAUAGUAUUGUCAAAGC seq id no: 442 seq id no: 724 EAM329 Oligo /5AmMC6/TCACCAAAACATGGAAGCACTTA UAAGUGCUUCCAUGUUUUGGUGA seq id no: 443 seq id no: 725 EAM330 Oligo /5AmMC6/GCTTCCAGTCGAGGATGTTTACA UGUAAACAUCCUCGACUGGAAGC seq id no: 444 seq id no: 726 EAM331 Oligo /5AmMC6/TCCAGTCAAGGATGTTTACA UGUAAACAUCCUUGACUGGA seq id no: 445 seq id no: 727 EAM332 Oligo /5AmMC6/CAGCTATGCCAGCATCTTGCCT AGGCAAGAUGCUGGCAUAGCUG seq id no: 446 seq id no: 728 EAM333 Oligo /5AmMC6/GCAACTTAGTAATGTGCAATA UAUUGCACAUUACUAAGUUGC seq id no: 447 seq id no: 729 EAM334 Oligo /5AmMC6/GAACCCACAATCCCTGGCTTA UAAGCCAGGGAUUGUGGGUUC seq id no: 448 seq id no: 730 EAM335 Oligo /5AmMC6/CAATCAGCTAATGACACTGCCT AGGCAGUGUCAUUAGCUGAUUG seq id no: 449 seq id no: 731 EAM336 Oligo /5AmMC6/GCAATCAGCTAACTACACTGCCT AGGCAGUGUAGUUAGCUGAUUGC seq id no: 450 seq id no: 732 EAM337 Oligo /5AmMC6/CTACCTGCACGAACAGCACTTTG CAAAGUGCUGUUCGUGCAGGUAG seq id no: 451 seq id no: 733 EAM338 Oligo /5AmMC6/TGCTCAATAAATACCCGTTGAA UUCAACGGGUAUUUAUUGAGCA seq id no: 452 seq id no: 734 EAM339 Oligo /5AmMC6/CGCTTGGTCGGTTCTTCGGGTG CACCCGUAGAACCGACCUUGCG seq id no: 453 seq id no: 735 EAM340 Oligo /5AmMC6/AGAAAGGCAGCAGGTCGTATAG CUAUACGACCUGCUGCCUUUCU seq id no: 454 seq id no: 736 EAM341 Oligo /5AmMC6/TACCTGCACTGTTAGCACTTTG CAAAGUGCUAACAGUGCAGGUA seq id no: 455 seq id no: 737 EAM342 Oligo /5AmMC6/CACATAGGAATGAAAAGCCATA UAUGGCUUUUCAUUCCUAUGUG seq id no: 456 seq id no: 738 EAM343 Oligo /5AmMC6/CCTCAAGGAGCCTCAGTCTAGT ACUAGACUGAGGCUCCUUGAGG seq id no: 457 seq id no: 739 EAM344 Oligo /5AmMC6/ACAAGTGCCCTCACTGCAGT ACUGCAGUGAGGGCACUUGU seq id no: 458 seq id no: 740 EAM345 Oligo /5AmMC6/TAAACGGAACCACTAGTGACTTA UAAGUCACUAGUGGUUCCGUUUA seq id no: 459 seq id no: 741 EAM346 Oligo /5AmMC6/AAAAAGTGCCCCCATAGTTTGAG CUCAAACUAUGGGGGCACUUUUU seq id no: 460 seq id no: 742 EAM347 Oligo /5AmMC6/GGCACACAAAGTGGAAGCACTTT AAAGUGCUUCCACUUUGUGUGCC seq id no: 461 seq id no: 743 EAM348 Oligo /5AmMC6/AGAGAGGGCCTCCACTTTGATG CAUCAAAGUGGAGGCCCUCUCU seq id no: 462 seq id no: 744 EAM349 Oligo /5AmMC6/ACACTCAAAACCTGGCGGCACTT AAGUGCCGCCAGGUUUUGAGUGU seq id no: 463 seq id no: 745 EAM350 Oligo /5AmMC6/CAAAAGAGCCCCCAGTTTGAGT ACUCAAACUGGGGGCUCUUUUG seq id no: 464 seq id no: 746 EAM351 Oligo /5AmMC6/ACACTACAAACTCTGCGGCACT AGUGCCGCAGAGUUUGUAGUGU seq id no: 465 seq id no: 747 EAM352 Oligo /5AmMC6/ACACACAAAAGGGAAGCACTTT AAAGUGCUUCCCUUUUGUGUGU seq id no: 466 seq id no: 748 EAM353 Oligo /5AmMC6/AGACTCAAAAGTAGTAGCACTTT AAAGUGCUACUACUUUUGAGUCU seq id no: 467 seq id no: 749 EAM354 Oligo /5AmMC6/CATGCACATGCACACATACAT AUGUAUGUGUGCAUGUGCAUG seq id no: 468 seq id no: 750 EAM355 Oligo /5AmMC6/GGAAGAACAGCCCTCCTCTGCC GGCAGAGGAGGGCUGUUCUUCC seq id no: 469 seq id no: 751 EAM356 Oligo /5AmMC6/GAAGAGAGCTTGCCCTTGCATA UAUGCAAGGGCAAGCUCUCUUC seq id no: 470 seq id no: 752 EAM357 Oligo /5AmMC6/TGTTGCTGCGCTTCTTGTTT AAACAUGAAGCGCUGCAACA seq id no: 471 seq id no: 753 EAM358 Oligo /5AmMC6/AGAGGTCGACCGTGTAATGTGC GCACAUUACACGGUCGACCUCU seq id no: 472 seq id no: 754 EAM359 Oligo /5AmMC6/CCAGCAGCACCTGGGGCAGT CCACUGCCCCAGGUGCUGCUGG seq id no: 473 seq id no: 755 EAM360 Oligo /5AmMC6/ACACTTACTGAGCACCTACTAGG CCUAGUAGGUGCUCAGUAAGUGU seq id no: 474 seq id no: 756 EAM361 Oligo /5AmMC6/ACTGGAGGAAGGGCCCAGAGG CCUCUGGGCCCUUCCUCCAGU seq id no: 475 seq id no: 757 EAM362 Oligo /5AmMC6/ACGGAAGGGCAGAGAGGGCCAG CUGGCCCUCUCUGCCCUUCCGU seq id no: 476 seq id no: 758 EAM363 Oligo /5AmMC6/AAAAAGGTTAGCTGGGTGTGTT AACACACCCAGCUAACCUUUUU seq id no: 477 seq id no: 759 EAM364 Oligo /5AmMC6/TCTCTGCTGGCCCTGTGCTTTGC GCAAAGCACAGGGCCUGCAGAGA seq id no: 478 seq id no: 760 EAM365 Oligo /5AmMC6/TTCTAGGATAGGCCCAGGGGC GCCCCUGGGCCUAUCCUAGAA seq id no: 479 seq id no: 761 EAM366 Oligo /5AmMC6/AAAGGCATCATATAGGAGCTGAA UUCAGCUCCUAUAUGAUGCCUUU seq id no: 480 seq id no: 762 EAM367 Oligo /5AmMC6/TCAACAAAATCACTGATGCTGGA UCCAGCAUCAGUGAUUUUGUUGA seq id no: 481 seq id no: 763 EAM368 Oligo /5AmMC6/TGAGCTCCTGGAGGACAGGGA UCCCUGUCCUCCAGGAGCUCA seq id no: 482 seq id no: 764 EAM369 Oligo /5AmMC6/GGCTATAAAGTAACTGAGACGGA UCCGUCUCAGUUACUUUAUAGCC seq id no: 483 seq id no: 765 EAM370 Oligo /5AmMC6/ACTGACCGACCGACCGATCGA UCGAUCGGUCGGUCGGUCAGU seq id no: 484 seq id no: 766 EAM371 Oligo /5AmMC6/GACGGGTGCGATTTCTGTGTGAGA UCUCACACAGAAAUCGCACCCGUC seq id no: 485 seq id no: 767 EAM372 Oligo /5AmMC6/ACAGTCAGGCTTTGGCTAGATCA UGAUCUAGCCAAAGCCUGACUGU seq id no: 486 seq id no: 768 EAM373 Oligo /5AmMC6/GCACTGGACTAGGGGTCAGCA UGCUGACCCCUAGUCCAGUGC seq id no: 487 seq id no: 769 EAM374 Oligo /5AmMC6/AGAGGCAGGCACTCGGGCAGA UGUCUGCCCGAGUGCCUGCCUCU seq id no: 488 seq id no: 770 EAM375 Oligo /5AmMC6/CAATCAGCTAATTACACTGCCTA UAGGCAGUGUAAUUAGCUGAUUG seq id no: 489 seq id no: 771 EAM376 Oligo /5AmMC6/GTGAAAGTGTATGGGCTTTGTG UUCACAAAGCCCAUACACUUUCAC seq id no: 490 seq id no: 772 EAM377 Oligo /5AmMC6/CAGGCTCAAAGGGCTCCTCAGG UCCCUGAGGAGCCCUUUGAGCCUG seq id no: 491 seq id no: 773 EAM378 Oligo /5AmMC6/AACAAAATCACAAGTCTTCCA UGGAAGACUUGUGAUUUUGUU seq id no: 492 seq id no: 774 EAM379 Oligo /5AmMC6/TTGCTTTTTGGGGTTTGGGCTT AAGCCCUUACCCCAAAAAGCAU seq id no: 493 seq id no: 775 EAM380 Oligo /5AmMC6/TGTCCGTGGTTCTTCCCTGTG UACCACAGGGUAGAACCACGGACA seq id no: 494 seq id no: 776 EAM381 Oligo /5AmMC6/TACTAGACTGTGAGCTCCTCGA UCGAGGAGCUCACAGUCUAGUA seq id no: 495 seq id no: 777 EAM382 Oligo /5AmMC6/TGTAAGTGCTCGTAATGCAGT ACUGCAUUACGAGCACUUACA seq id no: 496 seq id no: 778 EAM383 Oligo /5AmMC6/ACCCTCATGCCCCTCAAGG CCUUGAGGGGCAUGAGGGU seq id no: 497 seq id no: 779 EAM384 Oligo /5AmMC6/AAAAGTAACTAGCACACCAC GUGGUGUGCUAGUUACUUUU seq id no: 498 seq id no: 780 EAM385 Oligo /5AmMC6/ACATTTTTCGTTATTGCTCTT UCAAGAGCAAUAACGAAAAAUGU seq id no: 499 seq id no: 781 EAM386 Oligo /5AmMC6/AGACTAGATATGGAAGGGTGA UCACCCUUCCAUAUCUAGUCU seq id no: 500 seq id no: 782 EAM387 Oligo /5AmMC6/ACTGGGCACACGGAGGGAGA UCUCCCUCCGUGUGCCCAGU seq id no: 501 seq id no: 783 EAM388 Oligo /5AmMC6/ACGGTCAGGCTTTGGCTAGAT UGAUCUAGCCAAAGCCUGACCGU seq id no: 502 seq id no: 784 EAM389 Oligo /5AmMC6/AGAGGCAGGCACTCAGGCAGA UGUCUGCCUGAGUGCCUGCCUCU seq id no: 503 seq id no: 785 EAM390 Oligo /5AmMC6/TGGGCGACCCAGAGGGACA UGUCCCUCUGGGUCGCCCA seq id no: 504 seq id no: 786 EAM391 Oligo /5AmMC6/AGAGGTTAAGACAGCAGGGCTG CAGCCCUGCUGUCUUAACCUCU seq id no: 505 seq id no: 787 EAM392 Oligo /5AmMC6/TACTATGCAACCTACTACTCT AGAGUAGUAGGUUGCAUAGUA seq id no: 506 seq id no: 788 EAM393 Oligo /5AmMC6/TATGGCAGACTGTGATTTGTTG CAACAAAUCACAGUCUGCCAUA seq id no: 507 seq id no: 789 emc139 Oligo /5AmMC6/CGAAATGCGTCTCATACAAAATC NA seq id no: 508 seq id no: 790 EAM289 Oligo /5AmMC6/AACAAGCCCAGACCGCAAAAAG CUUUUUGCGGUCUGGGCUUGCU seq id no: 509 seq id no: 791 EAM283 Oligo /5AmMC6/AGGCAAAGGATGACAAAGGGAA UUCCCUUUGUCAUCCUUUGCCU seq id no: 510 seq id no: 792 PTG20210 Oligo /5AmC12/CATTGAGGCTCGCTGAGAGT GTGACTCTCAGCGAGCCTCAATGC seq id no: 511 seq id no: 793 MRC677 Oligo /5AmC12/GATGAAATCGGCTCCCGCAG- TGTCTGCGGGAGCCGATTTCATCA seq id no: 512 seq id no: 794 FVR506 Oligo /5AmC12/TGTATTCCTCGCCTGTCCAG TCCCTGGACAGGCGAGGAATACAG seq id no: 513 seq id no: 795 EAM104 Oligo /5AmMC6/TGGCATTCAGCGGGTGCCTTA TAAGGCACCCGCTGAATGCCA seq id no: 514 seq id no: 796 EAM106 Oligo /5AmMC6/TCACAAGTAAGGGTGTCAGGGA TCCCTGACACCCTTACTTGTGA seq id no: 515 seq id no: 797 EAM110 Oligo /5AmMC6/AACAACAAAATGAGTAGTCTTCCA TGGAAGACTACTCATTTTGTTGTT seq id no: 516 seq id no: 798 EAM1101 Oligo /5AmMC6/GTGGTAGCGCAGTGCGTAGAA TTCTACGCACTGCGCTACCAC seq id no: 517 seq id no: 799 EAM1102 Oligo /5AmMC6/GGTGATGCCCTGAATGTTGTC NA seq id no: 518 seq id no: 800 EAM1103 Oligo /5AmMC6/TGTCATGGATGACCTTGGCCA NA seq id no: 519 seq id no: 801 EAM1104 Oligo /5AmMC6/CTTTTGACATTGAAGGGAGCT NA seq id no: 520 seq id no: 802 EAM146 Oligo /5AmMC6/AACCATACAAGCTAGTACCTCA TGAGGTACTAGCTTGTATGGTT seq id no: 521 seq id no: 803 emc130 Oligo /5AmMC6/CTTGTACCAGTTATCTGCAA UUGCAGAUAACUGGUACAAG seq id no: 522 seq id no: 804 emc115 Oligo /5AmMC6/TTGTACGTTTACATGGAGGTC GACCUCCAUGUAAACGUACAA seq id no: 523 seq id no: 805 EAM148 Oligo /5AmMC6/AACCACACAAGCTAGTACCTCA TGAGGTACTAGCTTGTGTGGTT seq id no: 524 seq id no: 806 EAM138 Oligo /5AmMC6/CCGACCATGGGTGAAGACTGTTA TAACAGTCTTCACCCATGGTCGG seq id no: 525 seq id no: 807 EAM134 Oligo /5AmMC6/ACACCAATGGCGTAGGGGATGCG CGCATCCCCTACGCCATTGGTGT seq id no: 526 seq id no: 808 EAM395 Oligo /5AmMC6/CTGACTGACTGACTGACTGACTG CAGUCAGUCAGUCAGUCAGUCAG seq id no: 527 seq id no: 809 EAM149I Oligo /5AmMC6/GTCACTATTGTTGAGAACGTTGGCC NA seq id no: 528 seq id no: 810 EAM150I Oligo /5AmMC6/GTCACTATTGTAGAGAAGGTTGGCC NA seq id no: 529 seq id no: 811 EAM399 Oligo /5AmMC6/TTCAATTTCTGCCGCAAAAG UAUCUUUUGCGGCAGAAAUUGAA seq id no: 530 seq id no: 812 EAM400 Oligo /5AmMC6/GCTATCTGCTGCAACAGAATTT AAAUUCUGUUGCAGCAGAUAGC seq id no: 531 seq id no: 813 EAM401 Oligo /5AmMC6/GTGTGCTTACACACTTCCCGTTA UAACGGGAAGUGUGUAAGCACAC seq id no: 532 seq id no: 814 EAM402 Oligo /5AmMC6/TAGCTGGTTGAAGGGGACCAA UUGGUCCCCUUCAACCAGCUA seq id no: 533 seq id no: 815 EAM403 Oligo /5AmMC6/CCTCAAGGAGCTTCAGTCTAGT ACUAGACUGAAGCUCCUUGAGG seq id no: 534 seq id no: 816 EAM404 Oligo /5AmMC6/CCAACAACAGGAAACTACCTA UAGGUAGUUUCCUGUUGUUGG seq id no: 535 seq id no: 817 EAM405 Oligo /5AmMC6/CTACTAAAACATGGAAGCACTTA UAAGUGCUUCCAUGUUUUAGUAG seq id no: 536 seq id no: 818 EAM406 Oligo /5AmMC6/AGAAAGCACTTCCATGTTAAAGT ACUUUAACAUGGAAGUGCUUUCU seq id no: 537 seq id no: 819 EAM407 Oligo /5AmMC6/CCACTGAAACATGGAAGCACTTA UAAGUGCUUCCAUGUUUCAGUGG seq id no: 538 seq id no: 820 EAM408 Oligo /5AmMC6/CAGCAGGTACCCCCATGTTA UUUAACAUGGGGGUACCUGCUG seq id no: 539 seq id no: 821 EAM409 Oligo /5AmMC6/ACACTCAAACATGGAAGCACTTA UAAGUGCUUCCAUGUUUGAGUGU seq id no: 540 seq id no: 822 EAM410 Oligo /5AmMC6/ACTTACTGGACACCTACTAGG CCUAGUAGGUGUCCAGUAAGU seq id no: 541 seq id no: 823 EAM411 Oligo /5AmMC6/TCTCTGCTGGCCGTGTGCTT GCAAAGCACACGGCCUGCAGAGA seq id no: 542 seq id no: 824 EAM412 Oligo /5AmMC6/AAAGGCATCATATAGGAGCTGGA UCCAGCUCCUAUAUGAUGCCUUU seq id no: 543 seq id no: 825 EAM413 Oligo /5AmMC6/GCCCTGGACTAGGAGTCAGCA UGCUGACUCCUAGUCCAGGGC seq id no: 544 seq id no: 826 EAM414 Oligo /5AmMC6/AGAGGCAGGCATGCGGGCAG UGUCUGCCCGCAUGCCUGCCUCU seq id no: 545 seq id no: 827 EAM415 Oligo /5AmMC6/TCACCATTGCTAAAGTGCAATT AAUUGCACUUUAGCAAUGGUGA seq id no: 546 seq id no: 828 EAM416 Oligo /5AmMC6/AAACGTGGAATTTCCTCTATGT ACAUAGAGGAAAUUCCACGUUU seq id no: 547 seq id no: 829 EAM417 Oligo /5AmMC6/AAAGATCAACCATGTATTATT AAUAAUACAUGGUUGAUCUUU seq id no: 548 seq id no: 830 EAM418 Oligo /5AmMC6/CCAGGTTCCACCCCAGCAGG GCCUGCUGGGGUGGAACCUGG seq id no: 549 seq id no: 831 EAM419 Oligo /5AmMC6/ACACTCAAAAGATGGCGGCA GUGCCGCCAUCUUUUGAGUGU seq id no: 550 seq id no: 832 EAM420 Oligo /5AmMC6/ACGCTCAAATGTCGCAGCAC AAAGUGCUGCGACAUUUGAGCGU seq id no: 551 seq id no: 833 EAM421 Oligo /5AmMC6/ACACCCCAAAATCGAAGCAC GAAGUGCUUCGAUUUUGGGGUGU seq id no: 552 seq id no: 834 EAM422 Oligo /5AmMC6/GGAAAGCGCCCCCATTTTGA ACUCAAAAUGGGGGCGCUUUCC seq id no: 553 seq id no: 835 EAM423 Oligo /5AmMC6/CACTTATCAGGTTGTATTATAA UUAUAAUACAACCUGAUAAGUG seq id no: 554 seq id no: 836 EAM424 Oligo /5AmMC6/TAGCTGGTTGAAGGGGACCA UUGGUCCCCUUCAACCAGCUA seq id no: 555 seq id no: 837 EAM425 Oligo /5AmMC6/CCAACAACAGGAAACTACCTA UAGGUAGUUUCCUGUUGUUGG seq id no: 556 seq id no: 838 EAM426 Oligo /5AmMC6/GTCTGTCAAATCATAGGTCAT AUGACCUAUGAUUUGACAGAC seq id no: 557 seq id no: 839 EAM427 Oligo /5AmMC6/GGGGTTCACCGAGCAACATTC GAAUGUUGCUCGGUGAACCCCUU seq id no: 558 seq id no: 840 EAM428 Oligo /5AmMC6/CAGGCCATCTGTGTTATATT AAUAUAACACAGAUGGCCUGUU seq id no: 559 seq id no: 841 EAM429 Oligo /5AmMC6/AGTGGATGTTCCTCTATGAT AUCAUAGAGGAACAUCCACUUU seq id no: 560 seq id no: 842 EAM430 Oligo /5AmMC6/CGTGGATTTTCCTCTACGAT AUCGUAGAGGAAAAUCCACGUU seq id no: 561 seq id no: 843 EAM431 Oligo /5AmMC6/GAGGGTTAGTGGACCGTGTT AACACGGUCCACUAACCCUCAGU seq id no: 562 seq id no: 844 EAM432 Oligo /5AmMC6/GATGTGGACCATACTACATA UAUGUAGUAUGGUCCACAUCUU seq id no: 563 seq id no: 845 EAM433 Oligo /5AmMC6/GGCTAGTGGACCAGGTGAAG CUUCACCUGGUCCACUAGCCGU seq id no: 564 seq id no: 846 EAM396 Oligo /5AmMC6/AGCACGTCACTTCCACTAAGA UCUUAGUGGAAGUGACGUGCU seq id no: 565 seq id no: 847 EAM397 Oligo /5AmMC6/GCAAGGGCGAATGCAGAAAA UAUUUUCUGCAUUCGCCCUUGC seq id no: 566 seq id no: 848 EAM398 Oligo /5AmMC6/AACTCCGGGGCTGATCAGGT UAACCUGAUCAGCCCCGGAGUU seq id no: 567 seq id no: 849

TABLE 10b Set Set No. No. Probe ID Human Mouse Rat Other Control (V1) (V2) Usage EAM103 hsa- mmu- rno- 1 1 Used miR- miR- miR- 124a 124a 124a EAM105 hsa- mmu- rno- 1 1 Used miR- miR- miR- 125b 125b 125b EAM109 hsa- mmu- rno- 1 1 Used miR-7 miR-7 miR-7 EAM111 hsa-let- mmu- 1 1 Used 7g let-7g EAM115 hsa- mmu- rno- 1 1 Used miR-16 miR- miR- 16 16 EAM119 hsa- mmu- rno- 1 1 Used miR- miR- miR- 29b 29b 29b EAM121 hsa- mmu- rno- 1 1 Used miR- miR- miR- 99a 99a 99a EAM131 hsa- mmu- rno- 1 1 Used miR-92 miR- miR- 92 92 EAM139 hsa- mmu- rno- 1 1 Used miR- miR- miR- 146 146 146 EAM145 hsa-let- mmu- rno- 1 1 Used 7c let-7c let- 7c EAM152 hsa- mmu- 1 1 Used miR-9* miR- 9* EAM238 hsa- mmu- 1 1 Used miR-1 miR-1 EAM270 hsa- mmu- rno- 1 1 Used miR- miR- miR- 30b 30b 30b EAM159 hsa- mmu- rno- 1 1 Used miR- miR- miR- 130a 130a 130a EAM163 hsa- mmu- rno- 1 1 Used miR- miR- miR- 142-3p 142- 142- 3p 3p EAM171 hsa- mmu- rno- 1 1 Used miR- miR- miR- 137 137 137 EAM183 hsa-let- mmu- rno- 1 1 Used 7i let-7i let-7i EAM184 hsa- mmu- rno- 1 1 Used miR- miR- miR- 100 100 100 EAM186 hsa- 1 1 Used miR- 106a EAM189 hsa- mmu- rno- 1 1 Used miR- miR- miR- 10a 10a 10a EAM191 hsa- mmu- rno- 1 1 Used miR- miR- miR- 122a 122a 122a EAM192 hsa- mmu- rno- 1 1 Used miR- miR- miR- 126* 126* 126* EAM198 hsa- mmu- rno- 1 1 Used miR- miR- miR- 130b 130b 130b EAM202 hsa- mmu- rno- 1 1 Used miR- miR- miR- 134 134 134 EAM209 hsa- mmu- rno- 1 1 Used miR- miR- miR- 142-5p 142- 142- 5p 5p EAM221 mmu- 1 1 Used miR- 155 EAM223 hsa- mmu- rno- 1 1 Used miR- miR- miR- 15b 15b 15b EAM224 hsa- mmu- rno- 1 1 Used miR-17- miR- miR- 5p 17-5p 17 EAM225 hsa- mmu- rno- 1 1 Used miR-18 miR- miR- 18 18 EAM226 hsa- mmu- rno- 1 1 Used miR- miR- miR- 181a 181a 181a EAM227 hsa- mmu- rno- 1 1 Used miR- miR- miR- 181b 181b 181b EAM234 hsa- mmu- rno- 1 1 Used miR- miR- miR- 199a 199a 199a EAM235 hsa- 1 1 Used miR- 199b EAM236 hsa- mmu- rno- 1 1 Used miR- miR- miR- 19a 19a 19a EAM241 hsa- mmu- rno- 1 1 Used miR- miR- miR- 203 203 203 EAM242 hsa- mmu- rno- 1 1 Used miR- miR- miR- 204 204 204 EAM243 hsa- mmu- rno- 1 1 Used miR- miR- miR- 205 205 205 EAM245 hsa- mmu- rno- 1 1 Used miR- miR- miR- 210 210 210 EAM249 hsa- mmu- rno- 1 1 Used miR- miR- miR- 214 214 214 EAM254 hsa- mmu- rno- 1 3 Used miR- miR- miR- 219 219 219 EAM257 hsa- mmu- rno- 1 3 Used miR- miR- miR- 221 221 221 EAM258 hsa- mmu- rno- 1 3 Used miR- miR- miR- 222 222 222 EAM259 hsa- mmu- rno- 1 3 Used miR- miR- miR- 223 223 223 EAM273 hsa- mmu- rno- 1 3 Used miR-33 miR- miR- 33 33 EAM288 mmu- 1 3 Used miR- 10b EAM293 hsa- mmu- 1 3 Used miR- miR- 188 188 EAM297 hsa- mmu- rno- 1 3 Used miR- miR- miR- 193 193 193 EAM301 hsa- 1 3 Used miR- 198 EAM304 hsa- mmu- rno- 1 2 Used miR- miR- miR- 200a 200a 200a EAM306 mmu- 1 1 Used miR- 201 EAM307 mmu- 1 1 Used miR- 202 EAM308 hsa- mmu- rno- 1 1 Used miR- miR- miR- 206 206 206 EAM309 mmu- 1 1 Used miR- 207 EAM310 hsa- mmu- rno- 1 1 Used miR- miR- miR- 208 208 208 EAM247 hsa- mmu- rno- 1 1 Used miR- miR- miR- 212 212 212 EAM251 hsa- mmu- rno- 1 1 Used miR- miR- miR- 216 216 216 EAM253 hsa- mmu- rno- 1 1 Used miR- miR- miR- 218 218 218 EAM275 hsa- mmu- rno- 1 1 Used miR- miR- miR- 34a 34a 34a EAM246 hsa- 1 1 Used miR- 211 EAM250 hsa- 1 1 Used miR- 215 EAM252 hsa- 1 1 Used miR- 217 EAM305 mmu- 1 3 Used miR- 200b EAM303 hsa- mmu- 1 3 Used miR- miR- 199a* 199a* EAM300 hsa- 1 3 Used miR- 197 EAM299 hsa- mmu- rno- 1 3 Used miR- miR- miR- 195 195 195 EAM298 hsa- mmu- rno- 1 2 Used miR- miR- miR- 194 194 194 EAM296 hsa- mmu- rno- 1 2 Not Used, miR- miR- miR- high 191 191 191 background EAM295 hsa- mmu- rno- 1 2 Used miR- miR- miR- 190 190 190 EAM292 hsa- mmu- rno- 1 2 Used miR- miR- miR- 186 186 186 EAM112 Yes, 1 1 Not Used, Mismatch control feature EAM116 Yes, 1 1 Not Used, Mismatch control feature EAM120 Yes, 1 1 Not Used, Mismatch control feature EAM122 Yes, 1 1 Not Used, Mismatch control feature EAM132 Yes, 1 1 Not Used, Mismatch control feature EAM140 Yes, 1 1 Not Used, Mismatch control feature EAM282 mmu- 2 1 Used miR- 199b EAM281 mmu- rno- 2 1 Used miR- miR- 217 217 EAM280 hsa- mmu- rno- 2 1 Used miR- miR- miR- 30a-3p 30a- 30a- 3p 3p EAM279 hsa- mmu- rno- 2 1 Used miR- miR- miR- 29c 29c 29c EAM278 hsa- mmu- rno- 2 1 Used miR-98 miR- miR- 98 98 EAM277 hsa- mmu- rno- 2 3 Used miR-96 miR- miR- 96 96 EAM276 hsa- mmu- rno- 2 3 Used miR-9 miR-9 miR-9 EAM272 hsa- mmu- rno- 2 3 Used miR- miR- miR- 30d 30d 30d EAM271 hsa- mmu- rno- 2 3 Used miR- miR- miR- 30c 30c 30c EAM268 hsa- mmu- rno- 2 3 Used miR- miR- miR- 29a 29a 29a EAM264 hsa- mmu- rno- 2 3 Used miR- miR- miR- 27b 27b 27b EAM263 hsa- mmu- rno- 2 3 Used miR- miR- miR- 26a 26a 26a EAM262 hsa- mmu- rno- 2 3 Used miR-24 miR- miR- 24 24 EAM261 hsa- mmu- rno- 2 3 Used miR- miR- miR- 23b 23b 23b EAM260 hsa- mmu- rno- 2 3 Used miR- miR- miR- 23a 23a 23a EAM256 hsa- 2 3 Used miR- 220 EAM255 hsa- mmu- rno- 2 3 Used miR-22 miR- miR- 22 22 EAM248 hsa- mmu- rno- 2 3 Used miR- miR- miR- 213 213 213 EAM244 hsa- mmu- rno- 2 3 Used miR-21 miR- miR- 21 21 EAM240 hsa- mmu- rno- 2 3 Used miR-20 miR- miR- 20 20 EAM237 hsa- mmu- rno- 2 3 Used miR- miR- miR- 19b 19b 19b EAM233 hsa- mmu- rno- 2 3 Used miR- miR- miR- 196a 196a 196a EAM232 hsa- mmu- rno- 2 3 Used miR- miR- miR- 192 192 192 EAM231 hsa- mmu- rno- 2 3 Used miR- miR- miR- 187 187 187 EAM230 hsa- mmu- rno- 2 3 Used miR- miR- miR- 183 183 183 EAM229 hsa- mmu- 2 3 Used miR- miR- 182 182 EAM228 hsa- mmu- rno- 2 1 Used miR- miR- miR- 181c 181c 181c EAM222 hsa- mmu- 2 1 Used miR- miR- 15a 15a EAM220 hsa- mmu- rno- 2 3 Used miR- miR- miR- 154 154 154 EAM219 hsa- mmu- rno- 2 3 Used miR- miR- miR- 153 153 153 EAM218 hsa- mmu- rno- 2 3 Used miR- miR- miR- 152 152 152 EAM217 hsa- mmu- rno- 2 3 Used miR- miR- miR- 150 150 150 EAM216 hsa- mmu- 2 3 Used miR- miR- 149 149 EAM215 hsa- mmu- rno- 2 3 Used miR- miR- miR- 148b 148b 148b EAM214 hsa- mmu- 2 3 Used miR- miR- 148a 148a EAM212 hsa- mmu- rno- 2 3 Used miR- miR- miR- 145 145 145 EAM211 hsa- mmu- rno- 2 3 Used miR- miR- miR- 144 144 144 EAM210 hsa- mmu- rno- 2 3 Used miR- miR- miR- 143 143 143 EAM208 hsa- mmu- rno- 2 3 Used miR- miR- miR- 141 141 141 EAM207 hsa- mmu- rno- 2 3 Used miR- miR- miR- 140 140 140 EAM206 hsa- mmu- rno- 2 3 Used miR- miR- miR- 139 139 139 EAM205 hsa- mmu- rno- 2 3 Used miR- miR- miR- 138 138 138 EAM203 hsa- mmu- rno- 2 3 Used miR- miR- miR- 135a 135a 135a EAM200 hsa- mmu- rno- 2 3 Used miR- miR- miR- 133a 133a 133a EAM195 hsa- mmu- rno- 2 3 Used miR- miR- miR- 128b 128b 128b EAM194 hsa- mmu- rno- 2 3 Used miR- miR- miR- 128a 128a 128a EAM193 hsa- mmu- rno- 2 1 Used miR- miR- miR- 125a 125a 125a EAM190 hsa- rno- 2 1 Used miR- miR- 10b 10b EAM187 hsa- mmu- rno- 2 1 Used miR- miR- miR- 107 107 107 EAM185 hsa- mmu- rno- 2 1 Used miR- miR- miR- 103 103 103 EAM181 hsa-let- mmu- rno- 2 1 Used 7f let-7f let-7f EAM179 hsa-let- mmu- rno- 2 1 Used 7d let-7d let- 7d EAM177 mmu- rno- 2 1 Used miR- miR- 101b 101b EAM175 hsa- mmu- rno- 2 1 Used miR- miR- miR- 320 320 320 EAM168 hsa-let- mmu- rno- 2 1 Used 7e let-7e let- 7e EAM161 hsa- mmu- rno- 2 1 Used miR-28 miR- miR- 28 28 EAM160 hsa- mmu- rno- 2 1 Used miR- miR- miR- 26b 26b 26b EAM155 hsa- mmu- rno- 2 1 Used miR- miR- miR- 136 136 136 EAM153 hsa-let- mmu- rno- 2 1 Used 7a let-7a let- 7a EAM147 hsa-let- mmu- rno- 2 1 Used 7b let-7b let- 7b EAM137 hsa- mmu- rno- 2 1 Used miR- miR- miR- 132 132 132 EAM133 hsa- mmu- rno- 2 1 Used miR- miR- miR- 324-5p 324- 324- 5p 5p EAM311 hsa- mmu- rno- 2 2 Used miR- miR- miR- 101 101 101 EAM312 hsa- 2 2 Used miR- 105 EAM313 hsa- mmu- rno- 2 2 Used miR- miR- miR- 106b 106b 106b EAM314 hsa- mmu- rno- 2 2 Used miR- miR- miR- 126 126 126 EAM315 hsa- mmu- rno- 2 2 Used miR- miR- miR- 127 127 127 EAM316 hsa- 2 2 Used miR- 147 EAM317 hsa- 2 2 Used miR- 155 EAM318 hsa- 2 2 Used miR-17- 3p EAM319 hsa- 2 2 Used miR- 182* EAM320 hsa- mmu- 2 2 Used miR- miR- 189 189 EAM321 hsa- rno- 2 2 Used miR- miR- 200b 200b EAM291 hsa- mmu- rno- 2 2 Used miR- miR- miR- 185 185 185 EAM290 hsa- mmu- rno- 2 2 Used miR- miR- miR- 184 184 184 EAM322 hsa- mmu- rno- 3 2 Used miR- miR- miR- 200c 200c 200c EAM323 hsa- 3 2 Used miR- 224 EAM324 hsa- mmu- rno- 3 2 Used miR-25 miR- miR- 25 25 EAM325 hsa- mmu- rno- 3 2 Used miR- miR- miR- 27a 27a 27a EAM326 hsa- mmu- rno- 3 2 Used miR- miR- miR- 296 296 296 EAM327 hsa- mmu- rno- 3 2 Used miR- miR- miR- 299 299 299 EAM328 hsa- mmu- rno- 3 2 Used miR- miR- miR- 301 301 301 EAM329 hsa- mmu- 3 2 Used miR- miR- 302a 302 EAM330 hsa- mmu- rno- 3 2 Used miR- miR- miR- 30a-5p 30a- 30a- 5p 5p EAM331 hsa- mmu- rno- 3 2 Used miR- miR- miR- 30e 30e 30e EAM332 hsa- mmu- rno- 3 2 Used miR-31 miR- miR- 31 31 EAM333 hsa- mmu- rno- 3 2 Used miR-32 miR- miR- 32 32 EAM334 OLD_miR-321, 3 2 Used ARG_tRNA_FRAGMENT EAM335 hsa- 3 2 Used miR- 34b EAM336 hsa- mmu- rno- 3 2 Used miR- miR- miR- 34c 34c 34c EAM337 hsa- mmu- rno- 3 2 Used miR-93 miR- miR- 93 93 EAM338 hsa- 3 2 Used miR-95 EAM339 hsa- mmu- rno- 3 2 Used miR- miR- miR- 99b 99b 99b EAM340 mmu- rno- 3 2 Used let-7d* let- 7d* EAM341 mmu- 3 2 Used miR- 106a EAM342 hsa- mmu- rno- 3 2 Used miR- miR- miR- 135b 135b 135b EAM343 mmu- rno- 3 2 Used miR- miR- 151 151 EAM344 mmu- 3 2 Used miR- 17-3p EAM345 mmu- 3 2 Used miR- 224 EAM346 mmu- rno- 3 2 Used miR- miR- 290 290 EAM347 mmu- rno- 3 2 Used miR- miR- 291- 291- 3p 3p EAM348 mmu- rno- 3 2 Used miR- miR- 291- 291- 5p 5p EAM349 mmu- rno- 3 2 Used miR- miR- 292- 292- 3p 3p EAM350 mmu- rno- 3 2 Used miR- miR- 292- 292- 5p 5p EAM351 mmu- 3 2 Used miR- 293 EAM352 mmu- 3 2 Used miR- 294 EAM353 mmu- 3 2 Used miR- 295 EAM354 mmu- 3 2 Used miR- 297 EAM355 mmu- rno- 3 2 Used miR- miR- 298 298 EAM356 mmu- rno- 3 2 Used miR- miR- 300 300 EAM357 mmu- rno- 3 2 Used miR- miR- 322 322 EAM358 hsa- mmu- rno- 3 2 Used miR- miR- miR- 323 323 323 EAM359 hsa- mmu- rno- 3 2 Used miR- miR- miR- 324-3p 324- 324- 3p 3p EAM360 mmu- rno- 3 2 Used miR- miR- 325 325 EAM361 hsa- mmu- rno- 3 2 Used miR- miR- miR- 326 326 326 EAM362 hsa- mmu- rno- 3 2 Used miR- miR- miR- 328 328 328 EAM363 mmu- rno- 3 2 Used miR- miR- 329 329 EAM364 mmu- rno- 3 2 Used miR- miR- 330 330 EAM365 hsa- mmu- rno- 3 2 Used miR- miR- miR- 331 331 331 EAM366 mmu- rno- 3 2 Used miR- miR- 337 337 EAM367 hsa- mmu- rno- 3 2 Used miR- miR- miR- 338 338 338 EAM368 hsa- mmu- rno- 3 2 Used miR- miR- miR- 339 339 339 EAM369 hsa- mmu- rno- 3 2 Used miR- miR- miR- 340 340 340 EAM370 mmu- rno- 3 2 Used miR- miR- 341 341 EAM371 hsa- mmu- rno- 3 2 Used miR- miR- miR- 342 342 342 EAM372 mmu- 3 2 Used miR- 344 EAM373 mmu- rno- 3 2 Used miR- miR- 345 345 EAM374 mmu- 3 2 Used miR- 346 EAM375 mmu- rno- 3 2 Used miR- miR- 34b 34b EAM376 mmu- rno- 3 2 Used miR- miR- 350 350 EAM377 mmu- rno- 3 2 Used miR- miR- 351 351 EAM378 mmu- rno- 3 2 Used miR- miR- 7b 7b EAM379 rno- 3 2 Used miR- 129* EAM380 rno- 3 2 Used miR- 140* EAM381 rno- 3 2 Used miR- 151* EAM382 rno- 3 2 Used miR- 20* EAM383 rno- 3 2 Used miR- 327 EAM384 rno- 3 2 Used miR- 333 EAM385 hsa- mmu- rno- 3 2 Used miR- miR- miR- 335 335 335 EAM386 rno- 3 2 Used miR- 336 EAM387 rno- 3 2 Used miR- 343 EAM388 rno- 3 2 Used miR- 344 EAM389 rno- 3 2 Used miR- 346 EAM390 rno- 3 2 Used miR- 347 EAM391 rno- 3 2 Used miR- 349 EAM392 rno- 3 2 Used miR- 352 EAM393 rno- 3 2 Used miR- 7* emc139 Yes, Other 3 Not Not Used, control Used feature EAM289 hsa- mmu- rno- 3 1 Used miR- miR- miR- 129 129 129 EAM283 mmu- rno- 3 1 Used miR- miR- 211 211 PTG20210 Yes, post- 1, 2, 3 1, 2, 3 Not Used, control ctrl feature MRC677 Yes, Other 1, 2, 3 1, 2, 3 Not Used, control feature FVR506 Yes, post- 1, 2, 3 1, 2, 3 Not Used, control ctrl feature EAM104 Yes, 1, 2, 3 1 Not Used, control Mismatch feature EAM106 Yes, 1, 2, 3 1 Not Used, control Mismatch feature EAM110 Yes, 1, 2, 3 1 Not Used, control Mismatch feature EAM1101 Yes, 1, 2, 3 1 Not Used, control Mismatch feature EAM1102 Yes, Other 1, 2, 3 Not Not Used, control Used feature EAM1103 Yes, Other 1, 2, 3 Not Not Used, control Used feature EAM1104 Yes, Other 1, 2, 3 Not Not Used, control Used feature EAM146 Yes, 1, 2, 3 1 Not Used, control Mismatch feature emc130 Yes, Other 1, 2, 3 1, 2, 3 Not Used, control feature emc115 Yes, pre-ctrl 1, 2, 3 1, 2, 3 Not Used, control feature EAM148 Yes, 1, 2, 3 1 Not Used, control Mismatch feature EAM138 Yes, 1, 2, 3 1 Not Used, control Mismatch feature EAM134 Yes, 1, 2, 3 1 Not Used, control Mismatch feature EAM395 Yes, pre-ctrl 1, 2, 3 1, 2, 3 Not Used, control feature EAM149I Yes, Other 1, 2, 3 Not Not Used, control Used feature EAM150I Yes, Other 1, 2, 3 Not Not Used, control Used feature EAM399 ebv-miR-BHRF1-2 Not 3 Used only in ALL study Used EAM400 ebv-miR-BHRF1-2* Not 3 Used only in ALL study Used EAM401 ebv-miR-BHRF1-3 Not 3 Used only in ALL study Used EAM402 hsa- mmu- Not 3 Used only in ALL study miR- miR- Used 133b 133b EAM403 hsa- Not 3 Used only in ALL study miR- Used 151 EAM404 hsa- mmu- rno- Not 3 Used only in ALL study miR- miR- miR- Used 196b 196b 196b EAM405 hsa- Not 3 Used only in ALL study miR- Used 302b EAM406 hsa- Not 3 Used only in ALL study miR- Used 302b* EAM407 hsa- Not 3 Used only in ALL study miR- Used 302c EAM408 hsa- Not 3 Used only in ALL study miR- Used 302c* EAM409 hsa- Not 3 Used only in ALL study miR- Used 302d EAM410 hsa- Not 3 Used only in ALL study miR- Used 325 EAM411 hsa- Not 3 Used only in ALL study miR- Used 330 EAM412 hsa- Not 3 Used only in ALL study miR- Used 337 EAM413 hsa- Not 3 Used only in ALL study miR- Used 345 EAM414 hsa- Not 3 Used only in ALL study miR- Used 346 EAM415 hsa- Not 3 Used only in ALL study miR- Used 367 EAM416 hsa- Not 3 Used only in ALL study miR- Used 368 EAM417 hsa- Not 3 Used only in ALL study miR- Used 369 EAM418 hsa- mmu- Not 3 Used only in ALL study miR- miR- Used 370 370 EAM419 hsa- Not 3 Used only in ALL study miR- Used 371 EAM420 hsa- Not 3 Used only in ALL study miR- Used 372 EAM421 hsa- Not 3 Used only in ALL study miR- Used 373 EAM422 hsa- Not 3 Used only in ALL study miR- Used 373* EAM423 hsa- Not 3 Used only in ALL study miR- Used 374 EAM424 hsa- mmu- Not 3 Used only in ALL study miR- miR- Used 133b 133b EAM425 hsa- mmu- rno- Not 3 Used only in ALL study miR- miR- miR- Used 196b 196b 196b EAM426 mmu- Not 3 Used only in ALL study miR- Used 215 EAM427 mmu- Not 3 Used only in ALL study miR- Used 409 EAM428 mmu- Not 3 Used only in ALL study miR- Used 410 EAM429 mmu- Not 3 Used only in ALL study miR- Used 376b EAM430 mmu- Not 3 Used only in ALL study miR- Used 376a EAM431 mmu- Not 3 Used only in ALL study miR- Used 411 EAM432 mmu- Not 3 Used only in ALL study miR- Used 380- 3p EAM433 mmu- Not 3 Used only in ALL study miR- Used 412 EAM396 ebv-miR-BART1 Not 3 Used only in ALL study Used EAM397 ebv-miR-BART2 Not 3 Used only in ALL study Used EAM398 ebv-miR-BHRF1-1 Not 3 Used only in ALL study Used

TABLE 11 Oligonucleotide Sequences for Detection Specificity Experiment miRNA or Mutant Name Oligonucleotide Sequence (5′ to 3′) hsa-let-7g CTGGAATTCGCGGTTAAAACTGTACAAACTACTACCTCATTTAGTGAGGAATTCCGT (Seq ID No: 850) let-7-mutl CTGGAATTCGCGGTTAAATAACTGTAGAAAGTACTACCTCATTTAGTGAGGAATTCCGT (Seq ID No: 851) hsa-let-7c CTGGAATTCGCGGTTAAAAACCATACAACCTACTACCTCATTTAGTGAGGAATTCCGT (Seq ID No: 852) let-7-mut2 CTGGAATTCGCGGTTAAAAACCATACAAGCTAGTACCTCATTTAGTGAGGAATTCCGT (Seq ID No: 853) hsa-let-7b CTGGAATTCGCGGTTAAAAACCACACAACCTACTACCTCATTTAGTGAGGAATTCCGT (Seq ID No: 854) let-7-mut3 CTGGAATTCGCGGTTAAAAACCACACAAGCTAGTACCTCATTTAGTGAGGAATTCCGT (Seq ID No: 855) hsa-let-7a CTGGAATTCGCGGTTAAAAACTATACAACCTACTACCTCATTTAGTGAGGAATTCCGT (Seq ID No: 856) hsa-let-7e CTGGAATTCGCGGTTAAAACTATACAACCTCCTACCTCATTTAGTGAGGAATTCCGT (Seq ID No: 857) hsa-let-7d CTGGAATTCGCGGTTAAAACTATGCAACCTACTACCTCTTTTAGTGAGGAATTCCGT (Seq ID No: 858) hsa-let-7f CTGGAATTCGCGGTTAAAAACTATACAATCTACTACCTCATTTAGTGAGGAATTCCGT (Seq ID No: 858) hsa-let-7i CTGGAATTCGCGGTTAAAAGCACAAACTACTACCTCATTTAGTGAGGAATTCCGT (Seq ID No: 860)

TABLE 12 Alignment of Human let-7 miRNAs and  Mutant Sequences UGAGGUAGUAGUUUGUACAGU (Seq ID No: 861) hsa-let-7g UGAGGUAGUACUUUCUACAGUUA (Seq ID No: 862) let-7-mutl UGAGGUAGUAGGUUGUAUGGUU (Seq ID No: 863) hsa-let-7c UGAGGUACUAGCUUGUAUGGUU (Seq ID No: 864) let-7-mut2 UGAGGUAGUAGGUUGUGUGGUU (Seq ID No: 865) hsa-let-7b UGAGGUACUAGCUUGUGUGGUU (Seq ID No: 866) let-7-mut3 UGAGGUAGUAGGUUGUAUAGUU (Seq ID No: 867) hsa-let-7a UGAGGUAGGAGGUUGUAUAGU (Seq ID No: 868) hsa-let-7e AGAGGUAGUAGGUUGCAUAGU (Seq ID No: 869) hsa-let-7d UGAGGUAGUAGAUUGUAUAGUU (Seq ID No: 870) hsa-let-7f UGAGGUAGUAGUUUGUGCU (Seq ID No: 871) hsa-let-7i

TABLE 13 220 mRNA genes with transcription factor activity annotation Chip Probe Set ID Gene Title Hu6800 AB000468_at ring finger protein 4 Hu6800 D43642_at transcription factor-like 1 Hu6800 D83784_at pleiomorphic adenoma gene-like 2 Hu6800 D86479_at AE binding protein 1 Hu6800 D87673_at heat shock transcription factor 4 Hu6800 J03161_at serum response factor (c-fos serum response element- binding transcription factor) Hu6800 J03827_at nuclease sensitive element binding protein 1 Hu6800 L02785_at solute carrier family 26, member 3 Hu6800 L11672_at zinc finger protein 91 (HPF7, HTF10) Hu6800 L11672_r_at zinc finger protein 91 (HPF7, HTF10) Hu6800 L13203_at forkhead box I1 Hu6800 L13740_at nuclear receptor subfamily 4, group A, member 1 Hu6800 L17131_rna1_at high mobility group AT-hook 1 Hu6800 L20298_at core-binding factor, beta subunit Hu6800 L22342_at SP110 nuclear body protein Hu6800 L22454_at nuclear respiratory factor 1 Hu6800 L40904_at peroxisome proliferative activated receptor, gamma Hu6800 M14328_s_at enolase 1, (alpha) Hu6800 M16938_s_at homeo box C6 Hu6800 M19720_rna1_at v-myc myelocytomatosis viral oncogene homolog 1, lung carcinoma derived (avian) Hu6800 M23263_at androgen receptor (dihydrotestosterone receptor; testicular feminization; spinal and bulbar muscular atrophy; Kennedy disease) Hu6800 M24900_at thyroid hormone receptor, alpha (erythroblastic leukemia viral (v-erb-a) oncogene homolog, avian) /// nuclear receptor subfamily 1, group D, member 1 Hu6800 M25269_at ELK1, member of ETS oncogene family Hu6800 M31627_at X-box binding protein 1 Hu6800 M36542_s_at POU domain, class 2, transcription factor 2 Hu6800 M38258_at retinoic acid receptor, gamma Hu6800 M64673_at heat shock transcription factor 1 Hu6800 M65214_s_at transcription factor 3 (E2A immunoglobulin enhancer binding factors E12/E47) Hu6800 M68891_at GATA binding protein 2 Hu6800 M76732_s_at msh homeo box homolog 1 (Drosophila) Hu6800 M77698_at YY1 transcription factor Hu6800 M79462_at promyelocytic leukemia Hu6800 M79463_s_at promyelocytic leukemia Hu6800 M93650_at paired box gene 6 (aniridia, keratitis) Hu6800 M95929_at sideroflexin 3 Hu6800 M97676_at msh homeo box homolog 1 (Drosophila) Hu6800 M97935_s_at signal transducer and activator of transcription 1, 91 kDa Hu6800 M97936_at signal transducer and activator of transcription 1, 91 kDa Hu6800 M99701_at transcription elongation factor A (SII)-like 1 Hu6800 S81264_s_at T-box 2 Hu6800 U00968_at sterol regulatory element binding transcription factor 1 Hu6800 U11861_at maternal G10 transcript Hu6800 U18018_at ets variant gene 4 (E1A enhancer binding protein, E1AF) Hu6800 U20734_s_at jun B proto-oncogene Hu6800 U28687_at zinc finger protein 157 (HZF22) Hu6800 U29175_at SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4 Hu6800 U35048_at transforming growth factor beta 1 induced transcript 4 Hu6800 U36922_at forkhead box O1A (rhabdomyosarcoma) Hu6800 U39840_at forkhead box A1 Hu6800 U44755_at small nuclear RNA activating complex, polypeptide 2, 45 kDa Hu6800 U51003_s_at distal-less homeo box 2 Hu6800 U51127_at interferon regulatory factor 5 Hu6800 U53830_at interferon regulatory factor 7 Hu6800 U58681_at neurogenic differentiation 2 Hu6800 U63842_at neurogenin 1 Hu6800 U69126_s_at KH-type splicing regulatory protein (FUSE binding protein 2) Hu6800 U72649_at BTG family, member 2 Hu6800 U73843_at E74-like factor 3 (ets domain transcription factor, epithelial- specific) Hu6800 U76388_at nuclear receptor subfamily 5, group A, member 1 Hu6800 U81599_at homeo box B13 Hu6800 U81600_at paired related homeobox 2 Hu6800 U82759_at homeo box A9 Hu6800 U85193_at nuclear factor I/B Hu6800 U85658_at transcription factor AP-2 gamma (activating enhancer binding protein 2 gamma) Hu6800 U95040_at tripartite motif-containing 28 Hu6800 X03635_at estrogen receptor 1 Hu6800 X06614_at retinoic acid receptor, alpha Hu6800 X12794_at nuclear receptor subfamily 2, group F, member 6 Hu6800 X13293_at v-myb myeloblastosis viral oncogene homolog (avian)-like 2 Hu6800 X13810_s_at POU domain, class 2, transcription factor 2 Hu6800 X16316_at vav 1 oncogene Hu6800 X16665_at homeo box B2 Hu6800 X16706_at FOS-like antigen 2 Hu6800 X17360_rna1_at homeo box D4 Hu6800 X17651_at myogenin (myogenic factor 4) Hu6800 X51345_at jun B proto-oncogene Hu6800 X52541_at early growth response 1 Hu6800 X55005_rna1_at thyroid hormone receptor, alpha (erythroblastic leukemia viral (v-erb-a) oncogene homolog, avian) Hu6800 X55037_s_at GATA binding protein 3 Hu6800 X56681_s_at jun D proto-oncogene Hu6800 X58072_at GATA binding protein 3 Hu6800 X60003_s_at cAMP responsive element binding protein 1 Hu6800 X61755_rna1_s_at homeo box C5 Hu6800 X65463_at retinoid X receptor, beta Hu6800 X66079_at Spi-B transcription factor (Spi-1/PU.1 related) Hu6800 X68688_rna1_s_at zinc finger protein 11b (KOX 2) /// zinc finger protein 33a (KOX 31) Hu6800 X69699_at paired box gene 8 Hu6800 X70683_at SRY (sex determining region Y)-box 4 Hu6800 X72632_s_at thyroid hormone receptor, alpha (erythroblastic leukemia viral (v-erb-a) oncogene homolog, avian) /// nuclear receptor subfamily 1, group D, member 1 Hu6800 X78992_at zinc finger protein 36, C3H type-like 2 Hu6800 X85786_at regulatory factor X, 5 (influences HLA class II expression) Hu6800 X90824_s_at upstream transcription factor 2, c-fos interacting Hu6800 X93996_rna1_at myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, Drosophila); translocated to, 7 Hu6800 X96401_at MAX binding protein Hu6800 X96506_s_at DR1-associated protein 1 (negative cofactor 2 alpha) Hu6800 X99101_at estrogen receptor 2 (ER beta) Hu6800 Y08976_at FEV (ETS oncogene family) Hu6800 Z11899_s_at POU domain, class 5, transcription factor 1 Hu6800 Z17240_at high-mobility group box 2 Hu6800 Z22951_rna1_s_at — Hu6800 Z49825_s_at hepatocyte nuclear factor 4, alpha Hu6800 Z50781_at delta sleep inducing peptide, immunoreactor Hu6800 Z56281_at interferon regulatory factor 3 Hu35KsubA AA010750_at LAG1 longevity assurance homolog 2 (S. cerevisiae) Hu35KsubA AA036900_at FOS-like antigen 2 Hu35KsubA AA091017_at nuclear factor of activated T-cells 5, tonicity-responsive Hu35KsubA AA099501_at p66 alpha Hu35KsubA AA127183_s_at serologically defined colon cancer antigen 33 Hu35KsubA AA157520_at signal transducer and activator of transcription 5B Hu35KsubA AA287840_at Runt-related transcription factor 2 Hu35KsubA AA328684_at SLC2A4 regulator Hu35KsubA AA347664_at lymphoid enhancer-binding factor 1 Hu35KsubA AA355201_at SRY (sex determining region Y)-box 4 Hu35KsubA AA418098_at cAMP responsive element binding protein-like 2 Hu35KsubA AA424381_s_at Forkhead box C1 Hu35KsubA AA431268_at — Hu35KsubA AA436315_at forkhead box O3A Hu35KsubA AA456687_at nuclear factor I/A Hu35KsubA AA459542_s_at regulatory factor X-associated ankyrin-containing protein Hu35KsubA AA489299_at transcriptional adaptor 3 (NGG1 homolog, yeast)-like Hu35KsubA AA504413_at Solute carrier family 25, member 29 Hu35KsubA AB002302_at myeloid/lymphoid or mixed-lineage leukemia 4 Hu35KsubA AB002305_at aryl-hydrocarbon receptor nuclear translocator 2 Hu35KsubA AB004066_at basic helix-loop-helix domain containing, class B, 2 Hu35KsubA C02099_s_at methionine sulfoxide reductase B2 Hu35KsubA D45333_at prefoldin 1 Hu35KsubA D61676_at Pre-B-cell leukemia transcription factor 1 Hu35KsubA D82636_at CCR4-NOT transcription complex, subunit 7 Hu35KsubA H45647_at hairy/enhancer-of-split related with YRPW motif 1 Hu35KsubA IKAROS_at zinc finger protein, subfamily 1A, 1 (Ikaros) Hu35KsubA L07592_at peroxisome proliferative activated receptor, delta Hu35KsubA L13203_at forkhead box I1 Hu35KsubA L16794_s_at MADS box transcription enhancer factor 2, polypeptide D (myocyte enhancer factor 2D) Hu35KsubA L40904_at peroxisome proliferative activated receptor, gamma Hu35KsubA L41067_at nuclear factor of activated T-cells, cytoplasmic, calcineurin- dependent 3 Hu35KsubA M23263_at androgen receptor (dihydrotestosterone receptor; testicular feminization; spinal and bulbar muscular atrophy; Kennedy disease) Hu35KsubA M62626_s_at T-cell leukemia, homeobox 1 Hu35KsubA M79462_at promyelocytic leukemia Hu35KsubA M92299_s_at homeo box B5 Hu35KsubA M93650_at paired box gene 6 (aniridia, keratitis) Hu35KsubA M96577_s_at E2F transcription factor 1 Hu35KsubA M97676_at msh homeo box homolog 1 (Drosophila) Hu35KsubA N32724_at high-mobility group 20B Hu35KsubA N83192_at KIAA0669 gene product Hu35KsubA RC_AA029288_at zinc finger protein 83 (HPF1) Hu35KsubA RC_AA040699_at ELK3, ETS-domain protein (SRF accessory protein 2) Hu35KsubA RC_AA045545_at glucocorticoid modulatory element binding protein 2 Hu35KsubA RC_AA055932_at TAF5-like RNA polymerase II, p300/CBP-associated factor (PCAF)-associated factor, 65 kDa Hu35KsubA RC_AA065094_at trinucleotide repeat containing 4 Hu35KsubA RC_AA069549_at zinc finger protein 37a (KOX 21) Hu35KsubA RC_AA114866_s_at homeo box A11 Hu35KsubA RC_AA121121_at Huntingtin interacting protein 2 Hu35KsubA RC_AA135095_at high-mobility group 20B Hu35KsubA RC_AA136474_at Meis1, myeloid ecotropic viral integration site 1 homolog 2 (mouse) Hu35KsubA RC_AA150205_at Kruppel-like factor 7 (ubiquitous) Hu35KsubA RC_AA156112_at Krueppel-related zinc finger protein Hu35KsubA RC_AA156359_at TAR DNA binding protein Hu35KsubA RC_AA156792_at hairy/enhancer-of-split related with YRPW motif-like Hu35KsubA RC_AA235980_at transcription factor EB Hu35KsubA RC_AA252161_at p66 alpha Hu35KsubA RC_AA253429_at zinc finger protein 175 Hu35KsubA RC_AA256678_at CCR4-NOT transcription complex, subunit 7 Hu35KsubA RC_AA256680_at Nuclear factor I/B Hu35KsubA RC_AA280130_at checkpoint suppressor 1 Hu35KsubA RC_AA284143_at arginine-glutamic acid dipeptide (RE) repeats Hu35KsubA RC_AA286809_at upstream binding protein 1 (LBP-1a) Hu35KsubA RC_AA292717_at forkhead box P1 Hu35KsubA RC_AA347288_at growth arrest-specific 7 Hu35KsubA RC_AA379087_s_at apoptosis antagonizing transcription factor Hu35KsubA RC_AA393876_s_at nuclear receptor subfamily 2, group F, member 2 Hu35KsubA RC_AA419547_at E74-like factor 5 (ets domain transcription factor) Hu35KsubA RC_AA421050_at zinc finger protein 444 Hu35KsubA RC_AA425309_at Nuclear factor I/B Hu35KsubA RC_AA428024_at ubinuclein 1 Hu35KsubA RC_AA430032_at pituitary tumor-transforming 1 Hu35KsubA RC_AA431399_at arginine-glutamic acid dipeptide (RE) repeats Hu35KsubA RC_AA436608_at SATB family member 2 Hu35KsubA RC_AA443090_s_at interferon regulatory factor 7 Hu35KsubA RC_AA443962_at MYST histone acetyltransferase 2 Hu35KsubA RC_AA452256_at zinc finger protein 265 Hu35KsubA RC_AA456289_at nuclear factor I/A Hu35KsubA RC_AA456677_at zinc finger protein, subfamily 1A, 4 (Eos) Hu35KsubA RC_AA464251_at LOC440448 Hu35KsubA RC_AA476720_at nuclear factor of activated T-cells, cytoplasmic, calcineurin- dependent 1 Hu35KsubA RC_AA478590_at forkhead box O3A Hu35KsubA RC_AA478596_at zinc fingers and homeoboxes 2 Hu35KsubA RC_AA504110_at v-ets erythroblastosis virus E26 oncogene homolog 1 (avian) Hu35KsubA RC_AA504144_at CAMP responsive element binding protein 1 Hu35KsubA RC_AA504147_s_at Solute carrier family 25, member 29 Hu35KsubA RC_AA609017_s_at forkhead box O1A (rhabdomyosarcoma) Hu35KsubA RC_AA621179_at forkhead box J2 Hu35KsubA RC_AA621680_at Kruppel-like factor 4 (gut) Hu35KsubA RC_D59299_i_at myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, Drosophila); translocated to, 10 Hu35KsubA U09366_at zinc finger protein 133 (clone pHZ-13) Hu35KsubA U17163_at ets variant gene 1 Hu35KsubA U28687_at zinc finger protein 157 (HZF22) Hu35KsubA U33749_s_at thyroid transcription factor 1 Hu35KsubA U53831_s_at interferon regulatory factor 7 Hu35KsubA U62392_at zinc finger protein 193 Hu35KsubA U63824_at TEA domain family member 4 Hu35KsubA U76388_at nuclear receptor subfamily 5, group A, member 1 Hu35KsubA U81600_at paired related homeobox 2 Hu35KsubA U85707_at Meis1, myeloid ecotropic viral integration site 1 homolog (mouse) Hu35KsubA U88047_at AT rich interactive domain 3A (BRIGHT-like) Hu35KsubA U89995_at forkhead box E1 (thyroid transcription factor 2) Hu35KsubA W20276_f_at CG9886-like Hu35KsubA W26259_at forkhead box O3A Hu35KsubA W55861_at Myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, Drosophila) Hu35KsubA W67850_s_at TGFB-induced factor 2 (TALE family homeobox) Hu35KsubA X13403_s_at POU domain, class 2, transcription factor 1 Hu35KsubA X16666_s_at homeo box B1 Hu35KsubA X52402_s_at homeo box C5 Hu35KsubA X52560_s_at CCAAT/enhancer binding protein (C/EBP), beta Hu35KsubA X58431_rna2_s_at homeo box B6 Hu35KsubA X68688_rna1_s_at zinc finger protein 11b (KOX 2) /// zinc finger protein 33a (KOX 31) Hu35KsubA X70683_at SRY (sex determining region Y)-box 4 Hu35KsubA X99101_at estrogen receptor 2 (ER beta) Hu35KsubA X99350_rna1_at forkhead box J1 Hu35KsubA Y10746_at methyl-CpG binding domain protein 1 Hu35KsubA Z14077_s_at YY1 transcription factor

TABLE 14 Number of Training Samples Used to Build the Normal/Tumor Classifier Tissue Number of Normal Number of Tumor Colon 5 10 Kidney 3 5 Prostate 8 6 Uterus 9 10 Lung 4 6 Breast 3 6

TABLE 15 Normal/Tumor Makers Selected On the Training Set Bonferroni- Variance-thresholded Probe Description corrected p-value t-test score EAM159 hmr_miR-130a 0 10.984 EAM331 hmr_miR-30e 0 10.756 EAM311 hmr_miR-101 0 10.392 EAM299 hmr_miR-195 0 9.957 EAM314 hmr_miR-126 0 9.498 EAM300 h_miR-197 0 8.762 EAM181 hmr_let-7f 0 8.299 EAM380 r_miR-140* 0 8.238 EAM111 hm_let-7g 0 8.235 EAM381 r_miR-151* 0 8.198 EAM218 hmr_miR-152 0 8.180 EAM183 hmr_let-7i 0 8.098 EAM253 hmr_miR-218 0 8.077 EAM155 hmr_miR-136 0 8.058 EAM192 hmr_miR-126* 0 7.991 EAM222 hm_miR-15a 0 7.970 EAM161 hmr_miR-28 0 7.949 EAM184 hmr_miR-100 0 7.894 EAM271 hmr_miR-30c 0 7.848 EAM270 hmr_miR-30b 0 7.731 EAM303 hm_miR-199a* 0 7.519 EAM121 hmr_miR-99a 0 7.515 EAM392 r_miR-352 0 7.476 EAM255 hmr_miR-22 0 7.465 EAM249 hmr_miR-214 0 7.338 EAM160 hmr_miR-26b 0 7.313 EAM133 hmr_miR-324-5p 0 7.266 EAM238 hm_miR-1 0 7.259 EAM179 hmr_let-7d 0 7.235 EAM339 hmr_miR-99b 0 7.225 EAM185 hmr_miR-103 0 7.047 EAM168 hmr_let-7e 0 7.034 EAM200 hmr_miR-133a 0 6.959 EAM278 hmr_miR-98 0 6.952 EAM333 hmr_miR-32 0 6.951 EAM291 hmr_miR-185 0 6.910 EAM187 hmr_miR-107 0 6.879 EAM263 hmr_miR-26a 0 6.818 EAM261 hmr_miR-23b 0 6.814 EAM371 hmr_miR-342 0 6.743 EAM330 hmr_miR-30a-5p 0 6.717 EAM280 hmr_miR-30a-3p 0 6.662 EAM233 hmr_miR-196a 0 6.630 EAM292 hmr_miR-186 0 6.602 EAM115 hmr_miR-16 0 6.558 EAM272 hmr_miR-30d 0 6.516 EAM367 hmr_miR-338 0 6.428 EAM379 r_miR-129* 0 6.323 EAM193 hmr_miR-125a 0 6.222 EAM273 hmr_miR-33 0 6.209 EAM223 hmr_miR-15b 0 6.148 EAM105 hmr_miR-125b 0 6.111 EAM385 hmr_miR-335 0 6.011 EAM237 hmr_miR-19b 0 5.981 EAM320 hm_miR-189 0 5.938 EAM262 hmr_miR-24 0 5.909 EAM240 hmr_miR-20 0 5.908 EAM260 hmr_miR-23a 0 5.901 EAM297 hmr_miR-193 0 5.856 EAM236 hmr_miR-19a 0 5.789 EAM264 hmr_miR-27b 0 5.780 EAM205 hmr_miR-138 0 5.721 EAM234 hmr_miR-199a 0 5.718 EAM207 hmr_miR-140 0 5.561 EAM217 hmr_miR-150 0 5.531 EAM235 h_miR-199b 0 5.516 EAM190 hr_miR-10b 0 5.511 EAM282 m_miR-199b 0 5.483 EAM335 h_miR-34b 0 5.315 EAM288 m_miR-10b 0 5.291 EAM275 hmr_miR-34a 0 5.287 EAM195 hmr_miR-128b 0 5.253 EAM328 hmr_miR-301 0 5.203 EAM365 hmr_miR-331 0 5.191 EAM131 hmr_miR-92 0 5.155 EAM215 hmr_miR-148b 0 5.091 EAM325 hmr_miR-27a 0 5.090 EAM279 hmr_miR-29c 0 5.025 EAM369 hmr_miR-340 0 4.959 EAM354 m_miR-297 0 4.953 EAM119 hmr_miR-29b 0 4.937 EAM210 hmr_miR-143 0 4.908 EAM361 hmr_miR-326 0 4.790 EAM324 hmr_miR-25 0 4.764 EAM226 hmr_miR-181a 0 4.742 EAM343 mr_miR-151 0 4.740 EAM228 hmr_miR-181c 0 4.675 EAM366 mr_miR-337 0 4.661 EAM349 mr_miR-292-3p 0 4.652 EAM189 hmr_miR-10a 0 4.494 EAM355 mr_miR-298 0 4.446 EAM318 h_miR-17-3p 0 4.324 EAM387 r_miR-343 0 4.140 EAM363 mr_miR-329 0 4.118 EAM268 hmr_miR-29a 0 4.044 EAM175 hmr_miR-320 0 3.875 EAM212 hmr_miR-145 0 3.869 EAM378 mr_miR-7b 0 3.853 EAM281 mr_miR-217 0 3.670 EAM307 m_miR-202 0 3.625 EAM209 hmr_miR-142-5p 0 3.594 EAM163 hmr_miR-142-3p 0 3.545 EAM384 r_miR-333 0 3.410 EAM362 hmr_miR-328 0 3.356 EAM329 hm_miR-302a 0 3.348 EAM368 hmr_miR-339 0 3.007 EAM351 m_miR-293 0 2.852 EAM153 hmr_let-7a 0 2.818 EAM360 mr_miR-325 0 2.753 EAM145 hmr_let-7c 0 2.393 EAM348 mr_miR-291-5p 0 2.092 EAM298 hmr_miR-194 0 2.068 EAM250 h_miR-215 0 1.746 EAM229 hm_miR-182 0.005 −4.074 EAM224 hmr_miR-17-5p 0.005 4.875 EAM341 m_miR-106a 0.005 4.185 EAM242 hmr_miR-204 0.005 3.457 EAM295 hmr_miR-190 0.005 3.186 EAM353 m_miR-295 0.005 2.916 EAM246 h_miR-211 0.005 2.663 EAM248 hmr_miR-213 0.01 3.369 EAM186 h_miR-106a 0.01 4.650 EAM137 hmr_miR-132 0.01 3.388 EAM258 hmr_miR-222 0.015 4.257 EAM230 hmr_miR-183 0.02 −3.977 EAM364 mr_miR-330 0.02 3.982 EAM206 hmr_miR-139 0.02 3.761 EAM327 hmr_miR-299 0.025 2.353 EAM232 hmr_miR-192 0.04 1.065 EAM257 hmr_miR-221 0.04 4.321 EAM216 hm_miR-149 0.04 3.711

TABLE 16 Prediction results of mouse lung samples Field Description SAMPLE Sample name MAL Malignancy status (Normal/Tumor) PRED-MAL Predicted Malignancy status (Normal/Tumor). Prediction performed by kNN (k = 3) using a training set of 75 samples CORRECT? Is the prediction correct? Test set: 12 mouse lung samples SAMPLE MAL PRED-MAL CORRECT? N_MLUNG_1 Normal Normal Yes N_MLUNG_2 Normal Normal Yes N_MLUNG_3 Normal Normal Yes N_MLUNG_4 Normal Normal Yes N_MLUNG_5 Normal Normal Yes T_MLUNG_1 Tumor Tumor Yes T_MLUNG_2 Tumor Tumor Yes T_MLUNG_3 Tumor Tumor Yes T_MLUNG_4 Tumor Tumor Yes T_MLUNG_5 Tumor Tumor Yes T_MLUNG_6 Tumor Tumor Yes T_MLUNG_7 Tumor Tumor Yes

TABLE 20 Training and prediction results of poorly differentiated tumors Field Description Tissue Type Tissue type; COLON for colon, PAN for pancreas, KID for kidney, BLDR for bladder, PROST for prostate, OVARY for ovary, UT for uterus, LUNG for human lung, MESO for mesothelioma, MELA for melanoma, BRST for breast TT Tissue type code; 1 for stomach, 2 for colon, 3 for pancreas, 4 for liver, 5 for kidney, 6 for bladder, 7 for prostate, 8 for ovary, 9 for uterus, 10 for human lung, 11 for mesothelioma, 12 for melanoma, 13 for breast, 14 for brain, 19 for B cell ALL, 20 for T cell ALL, 21 for follicular cleaved lymphoma, 22 for large B cell lymphoma, 23 for mycosis fungoidis, 24 for myoloid, 26 for mouse lung # of features Number of features selected by the leave-one-out cross-validation procedure SIG The selected σ used in the PNN is SIG times the median nearest neighbor distance (see Supplementary Methods) NS Number of samples of the specific tissue-type in the training set NERR Number of leave-one-out errors for the selected parameters (Number of features and SIG) (=FP + FN) FP Number of false positives (incorrectly predicted to belong to the specific tissue type) FN Number of false negatives (incorrectly predicted to not belong to the specific tissue type) LL Log-likelihood of the selected parameters (Number of features and SIG) TRUE Code of true tisue-type of the test sample (see description of TT) PRED Predicted tissue-type (see description of TT for code explanation) PROB The PNN's posterior probability to belong to the class CORR Is this a correct classification; 1 for correct and 0 for incorrect miRNA Data Training set: 68 samples, 11 tissue-types Tissue # of Type TT features SIG NS NERR FP FN LL COLON 2 16 1 7 2 1 1 −0.075482 PAN 3 30 1.5 8 2 0 2 −0.175494 KID 5 28 1.5 4 1 0 1 −0.047266 BLDR 6 10 1 6 3 0 3 −0.901522 PROST 7 10 2.5 6 1 0 1 −0.181041 OVARY 8 18 1 5 2 1 1 −0.074861 UT 9 30 1 10 3 1 2 −0.151537 LUNG 10 28 1 5 1 0 1 −0.086595 MESO 11 30 1 8 0 0 0 −0.026769 MELA 12 18 1 3 0 0 0 −0.010752 BRST 13 22 1 6 2 1 1 −0.072847 Test set: 17 samples, 4 tissue-types SAMPLE PDT_COLON_1 PDT_OVARY_1 PDT_OVARY_2 PDT_OVARY_3 PDT_LUNG_1 PDT_LUNG_2 TRUE 2 8 8 8 10 10 PRED 2 8 8 8 2 10 PROB 0.95 0.838 0.823 0.929 0.312 0.207 CORR 1 1 1 1 0 1 SAMPLE PDT_LUNG_3 PDT_LUNG_4 PDT_LUNG_5 PDT_LUNG_6 PDT_LUNG_7 PDT_LUNG_8 TRUE 10 10 10 10 10 10 PRED 13 7 10 10 13 10 PROB 0.161 0.128 0.229 0.345 0.377 0.299 CORR 0 0 1 1 0 1 SAMPLE PDT_BRST_1 PDT_BRST_2 PDT_BRST_3 PDT_BRST_4 PDT_BRST_5 TRUE 13 13 13 13 13 PRED 13 13 13 9 13 PROB 0.905 0.479 0.552 0.476 0.773 CORR 1 1 1 0 1 Test set: Posterior probability matrix Tissue SAMPLE Type PDT_COLON_1 PDT_OVARY_1 PDT_OVARY_2 PDT_OVARY_3 PDT_LUNG_1 PDT_LUNG_2 COLON 0.95* 0 0 0 0.242 0 PAN 0.069 0.012 0.011 0.004 0.034 0.003 KID 0 0 0 0 0.02 0 BLDR 0 0 0 0 0 0 PROST 0 0.003 0.001 0 0 0.078 OVARY 0 0.838* 0.823* 0.929* 0.03 0 UT 0 0.342 0.193 0.225 0.312 0 (1) LUNG 0 0 0 0 0 0.207* MESO 0 0 0 0 0 0.002 MELA 0 0 0 0 0 0 BRST 0 0.001 0 0 0.001 0 Tissue SAMPLE Type PDT_LUNG_3 PDT_LUNG_4 PDT_LUNG_5 PDT_LUNG_6 PDT_LUNG_7 PDT_LUNG_8 COLON 0 0 0 0.247 0 0 PAN 0 0.001 0.004 0.152 0.006 0 KID 0 0 0 0 0 0 BLDR 0.01 0 0 0 0 0 PROST 0 0.128 0.011 0 0.048 0.03 OVARY 0.001 0.121 0.025 0 0.003 0.001 UT 0.029 0 0.012 0.001 0 0.002 LUNG 0.002 0.11 0.229* 0.345* 0.377 0.299* MESO 0 0 0 0 0 0 MELA 0 0 0 0 0 0 BRST 0.161 0.074 0 0.02 0.659 0.149 Tissue SAMPLE Type PDT_BRST_1 PDT_BRST_2 PDT_BRST_3 PDT_BRST_4 PDT_BRST_5 COLON 0 0 0 0 0 PAN 0.003 0.011 0 0.004 0.007 KID 0 0 0 0 0 BLDR 0.002 0.001 0.077 0.006 0 PROST 0.001 0.003 0.001 0 0.003 OVARY 0 0 0.13 0.009 0 UT 0.003 0 0.004 0.476 0.005 LUNG 0.017 0.035 0 0 0.277 MESO 0 0 0 0 0 MELA 0 0 0 0 0 BRST 0.905* 0.479* 0.552* 0 0.773* mRNA Data Training set: 68 samples, 11 tissue-types Tissue # of Type TT features SIG NS NERR FP FN LL COLON 2 18 1.5 7 0 0 0 −0.033006 PAN 3 14 4 8 1 0 1 −0.15038 KID 5 26 4 4 3 0 3 −0.16908 BLDR 6 10 1 6 5 1 4 −1.852998 PROST 7 30 4 6 1 0 1 −0.288903 OVARY 8 14 4 5 4 2 2 −0.2573 UT 9 20 3 10 2 0 2 −0.228232 LUNG 10 30 2.5 5 2 1 1 −0.119642 MESO 11 24 1.5 8 1 0 1 −0.095081 MELA 12 14 4 3 1 0 1 −0.164286 BRST 13 22 1 6 3 1 2 −0.450012 Test set: 17 samples, 4 tissue-types SAMPLE PDT_COLON_1 PDT_OVARY_1 PDT_OVARY_2 PDT_OVARY_3 PDT_LUNG_1 PDT_LUNG_2 TRUE 2 8 8 8 10 10 PRED 7 5 9 8 8 6 PROB 0.013 1 0.376 0.76 0.229 0.128 CORR 0 0 0 1 0 0 SAMPLE PDT_LUNG_3 PDT_LUNG_4 PDT_LUNG_5 PDT_LUNG_6 PDT_LUNG_7 PDT_LUNG_8 TRUE 10 10 10 10 10 10 PRED 6 3 8 8 8 6 PROB 0.022 0.102 0.305 0.014 0.091 0.173 CORR 0 0 0 0 0 0 SAMPLE PDT_BRST_1 PDT_BRST_2 PDT_BRST_3 PDT_BRST_4 PDT_BRST_5 TRUE 13 13 13 13 13 PRED 9 8 8 6 3 PROB 0.133 0.362 0.301 0.05 0.027 CORR 0 0 0 0 0 Test set: Posterior probability matrix Tissue SAMPLE Type PDT_COLON_1 PDT_OVARY_1 PDT_OVARY_2 PDT_OVARY_3 PDT_LUNG_1 PDT_LUNG_2 COLON 0 0 0 0 0 0 PAN 0.012 0.019 0.005 0.002 0.027 0.024 KID 0 1 0 0 0 0 BLDR 0.001 0.166 0.001 0.003 0.191 0.128 PROST 0.013 0.006 0.012 0.081 0.006 0.007 OVARY 0 0 0.244 0.76* 0.229 0.072 UT 0 0 0.376 0.084 0.074 0.007 LUNG 0.001 0 0.261 0 0 0 MESO 0 0.01 0.007 0.001 0.004 0 MELA 0 0 0 0 0 0 BRST 0 0.142 0 0 0.018 0 Tissue SAMPLE Type PDT_LUNG_3 PDT_LUNG_4 PDT_LUNG_5 PDT_LUNG_6 PDT_LUNG_7 PDT_LUNG_8 COLON 0 0 0 0 0 0 PAN 0.004 0.102 0.016 0.009 0.011 0.03 KID 0 0 0 0 0 0 BLDR 0.022 0.041 0.059 0.001 0.057 0.173 PROST 0.015 0.002 0.028 0.005 0.005 0.005 OVARY 0.006 0.062 0.305 0.014 0.091 0.055 UT 0.013 0.038 0.05 0.002 0.009 0.012 LUNG 0.005 0 0 0.001 0.003 0 MESO 0.003 0.006 0.024 0.01 0.002 0.001 MELA 0 0 0 0 0 0 BRST 0 0 0 0 0 0 Tissue SAMPLE Type PDT_BRST_1 PDT_BRST_2 PDT_BRST_3 PDT_BRST_4 PDT_BRST_5 COLON 0 0 0 0 0 PAN 0.016 0.026 0.019 0.021 0.027 KID 0 0 0 0 0 BLDR 0.014 0.044 0.237 0.05 0.003 PROST 0.006 0.025 0.001 0.003 0.021 OVARY 0.01 0.362 0.301 0 0 UT 0.133 0.01 0.036 0.011 0.001 LUNG 0 0.001 0.002 0 0 MESO 0.044 0 0.002 0.007 0.01 MELA 0 0 0 0 0 BRST 0 0.001 0.253 0 0 italic predicted bold True type *predicted correctly 

We claim:
 1. A solution-based method for determining the expression level of a population of target nucleic acids, comprising: a) providing in solution a population of target-specific bead sets, wherein each target-specific bead set is individually detectable and comprises a capture probe which corresponds to an individual target nucleic acid referred to as an individual bead set; b) hybridizing in solution the population of target-specific bead sets with a population of molecules that can contain a population of detectable target molecules, wherein each target nucleic acid has been transformed into a corresponding detectable target molecule which will specifically bind to its corresponding individual target-specific bead set; and c) screening in solution for detectable target molecules hybridized to target-specific beads to determine the expression level of the population of target nucleic acids.
 2. The method of claim 1, wherein the population of target-specific bead sets comprises at least 5 individual bead sets that can bind with a corresponding set of target nucleic acids.
 3. The method of claim 1, wherein the population of target-specific beads comprises at least 100 individual bead sets that can bind with a corresponding set of target nucleic acids.
 4. The method of claim 1, wherein the population of target nucleic acids is a population of mRNAs.
 5. The method of claim 1, wherein the population of target nucleic acids is a population of mRNAs and wherein each mRNA has been transformed into a corresponding detectable target molecule by a process comprising: a) reverse transcribing the mRNA target nucleic acid to generate a cDNA; b) contacting the cDNA with an upstream probe and a downstream probe, wherein the upstream probe comprises a universal upstream sequence and an upstream target-specific sequence, and the downstream probe comprises a universal downstream sequence and a downstream target-specific sequence, such that when the upstream probe and the downstream probe are both hybridized to the cDNA the two probes are capable of being ligated; c) ligating said cDNA contacted with said upstream and downstream probes to generate ligation complexes; and d) amplifying said ligation complexes with a pair of universal primers comprising a universal upstream primer and a universal downstream primer, wherein the universal upstream primer is complementary to the universal upstream sequence and the universal downstream primer is complementary to the universal downstream sequence, wherein at least one of the pair of universal primers is detectably labeled, wherein the product of the amplification is detectably labeled, thereby generating a detectable target molecule which corresponds to the target nucleic acid.
 6. The method of claim 1, wherein the population of target nucleic acids is a population of mRNAs, wherein each mRNA has been transformed into a corresponding detectable target molecule by a process comprising: a) reverse transcribing the mRNA target nucleic acid to generate a cDNA; b) contacting the cDNA with an upstream probe and a downstream probe, wherein the upstream probe comprises a universal upstream sequence and an upstream target-specific sequence, and the downstream probe comprises a universal downstream sequence and a downstream target-specific sequence, such that when the upstream probe and the downstream probe are both hybridized to the cDNA the two probes are capable of being ligated; c) ligating said cDNA contacted with said upstream and downstream probes to generate ligation complexes; and d) amplifying said ligation complexes with a pair of universal primers comprising a universal upstream primer and a universal downstream primer, wherein the universal upstream primer is complementary to the universal upstream sequence and the universal downstream primer is complementary to the universal downstream sequence, wherein at least one of the pair of universal primers is detectably labeled, wherein the product of the amplification is detectably labeled, thereby generating a detectable target molecule which corresponds to the target nucleic acid, wherein either the upstream probe further comprises an amplicon tag between the universal sequence and the target-specific sequence or the downstream probe further comprises an amplicon tag between the universal sequence and the target-specific sequence, wherein the amplicon tag comprises a nucleic acid sequence that is complementary to the sequence of the capture probe of the bead set.
 7. A method of identifying an expression signature associated with the presence or risk of cancer, infection, cellular disorder, or response to treatment comprising: a) isolating cells from a group of individuals with said cancer, infection, cellular disorder, or response to treatment, and determining the expression levels of a group of genes; b) isolating cells from a group of individuals without said cancer, infection, cellular disorder, or response to treatment, and determining the expression levels of said group of genes; and c) identifying differentially expressed genes from said group of genes which are together indicative of the presence or risk of cancer, infection, cellular disorder, or response to treatment in an individual, thereby identifying an expression signature associated with the presence or risk of cancer, infection, cellular disorder, or response to treatment, wherein the expression levels of the group of genes is determined using the method of claim 1 and the population of target nucleic acids are mRNAs.
 8. The method of claim 1, wherein the population of target nucleic acids is a population of microRNAs.
 9. A method of identifying an expression signature associated with the presence or risk of cancer, infection, cellular disorder, or response to treatment comprising: a) isolating cells from a group of individuals with said cancer, infection, cellular disorder, or response to treatment, and determining the expression levels of a group of genes; b) isolating cells from a group of individuals without said cancer, infection, cellular disorder, or response to treatment, and determining the expression levels of said group of genes; and c) identifying differentially expressed genes from said group of genes which are together indicative of the presence or risk of cancer, infection, cellular disorder, or response to treatment in an individual, thereby identifying an expression signature associated with the presence or risk of cancer, infection, cellular disorder, or response to treatment, wherein the expression levels of the group of genes is determined using the method of claim 1, wherein the population of target nucleic acids is a population of microRNAs and, wherein the expression signature comprises at least 5 genes.
 10. The method of claim 1, wherein the population of target nucleic acids is a population of microRNAs and wherein each microRNA has been transformed into a corresponding detectable target molecule by a process comprising: a) ligating at least one adaptor to the microRNA, generating an adaptor-microRNA molecule; b) detectably labeling said adaptor-microRNA molecule, thereby generating a detectable target molecule which corresponds to the target nucleic acid.
 11. The method of claim 1, wherein the population of target nucleic acids is a population of microRNAs and wherein each microRNA has been transformed into a corresponding detectable target molecule by a process comprising: a) ligating at least one adaptor to the microRNA, generating an adaptor-microRNA molecule; b) detectably labeling said adaptor-microRNA molecule, thereby generating a detectable target molecule which corresponds to the target nucleic acid, wherein the adaptor-microRNA is detectably labeled by reverse transcription using the adaptor-microRNA as a template for polymerase chain reaction, wherein a pair of primers is used in said polymerase chain reaction, and wherein at least one of said primers is detectably labeled.
 12. A method of screening for the presence of malignant cells in a test sample comprising: a) determining the level of expression of a group of microRNAs in the test sample, and b) comparing the level of expression of a group of microRNAs between the test sample and a corresponding reference sample, wherein a lower level of expression of the group of microRNAs in the test sample compared to the reference sample is indicative of the test sample containing malignant cells.
 13. The method of claim 12, wherein the reference sample is known to express a predetermined expression signature indicative of the presence of malignancy, infection, or cellular disorder, and the similarity of the expression signature of the test sample to the predetermined expression signature of the reference sample indicates the presence of malignant cells, infected cells, or cellular disorder, in the test sample.
 14. The method of claim 12, wherein the group of microRNAs comprises at least 5 microRNAs.
 15. The method of claim 12, wherein the test sample is isolated from an individual at risk of or suspected of having cancer.
 16. A method of classifying a tumor sample comprising: a) determining the expression pattern of a group of microRNAs in a tumor sample of unknown tissue origin, generating a tumor sample profile; b) providing a model of tumor origin microRNA expression patterns based on a dataset of the expression of microRNAs of tumors of known origin; and c) comparing the tumor sample profile to the model to determine which tumors of known origin the sample most closely resembles, thereby classifying the tissue origin of the tumor sample.
 17. A method of classifying a tumor sample comprising: a) determining the expression pattern of a group of microRNAs in a tumor sample of unknown tissue origin, generating a tumor sample profile; b) providing a model of tumor origin microRNA expression patterns based on a dataset of the expression of microRNAs of tumors of known origin; and c) comparing the tumor sample profile to the model to determine which tumors of known origin the sample most closely resembles, thereby classifying the tissue origin of the tumor sample, wherein the expression pattern of the group of microRNAs is determined using the methods of claim 1, wherein each target nucleic acid is a microRNA which has been transformed into a corresponding detectable target molecule by a process comprising: d) ligating at least one adaptor to the microRNA, generating an adaptor-microRNA molecule; e) detectably labeling said adaptor-microRNA molecule, thereby generating a detectable target molecule which corresponds to the target nucleic acid.
 18. A method for identifying an active compound or molecule, comprising: contacting cells with a plurality of compounds or molecules, determining the expression of a set of marker genes present in the cells using the method of claim 1, and scoring the expression of the marker genes to identify a cellular phenotype, the presence of a specific cellular phenotype being indicative of an active compound or molecule.
 19. A method for identifying an active compound or molecule, comprising: contacting cells with a plurality of compounds or molecules, determining the expression of a set of marker genes present in the cells using the method of claim 1, and scoring the expression of the marker genes to identify a cellular phenotype, the presence of a specific cellular phenotype being indicative of an active compound or molecule, wherein the set of marker genes comprises genes which encode microRNAs.
 20. A method for identifying an active compound or molecule, comprising: contacting cells with a plurality of compounds or molecules, determining the expression of a set of marker genes present in the cells using the method of claim 1, and scoring the expression of the marker genes to identify a cellular phenotype, the presence of a specific cellular phenotype being indicative of an active compound or molecule, wherein the set of marker genes comprises genes which encode messenger RNAs.
 21. A kit for determining in solution the expression level of a population of target nucleic acids, wherein said kit comprises: a) a population of detectable bead sets, wherein each target-specific bead set is individually detectable and is capable of being coupled to a capture probe which corresponds to an individual target nucleic acid of interest; b) components for transforming a target nucleic acid of interest into a corresponding detectable target molecule which will specifically bind to its corresponding individual target-specific bead set c) capture probes capable of specifically hybridizing to at least 10 different microRNAs or at least 10 different mRNAs.
 22. The kit of claim 21, wherein the population of target nucleic acids comprises mRNAs, wherein the kit further comprises a) components for reverse transcribing the mRNA to generate cDNA; b) upstream and downstream probes, wherein the upstream probe comprises a universal upstream sequence and an upstream target-specific sequence, and the downstream probe comprises a universal downstream sequence and a downstream target-specific sequence, such that when the upstream probe and the downstream probe are both hybridized to the cDNA the two probes are capable of being ligated; c) components for ligating DNA; d) a pair of universal primers; and e) components for amplifying DNA.
 23. The kit of claim 21, wherein the population of target nucleic acids comprises microRNAs, wherein the kit further comprises a) adaptors; b) components for ligating the microRNAs to the adaptors; c) components for reverse transcribing the microRNA to generate cDNA; d) a pair of universal primers; and e) components for amplifying DNA.
 24. The kit of claim 21, further comprising a polymerase and nucleotide bases.
 25. The kit of claim 21, further comprising a plurality of detectable labels. 